Extracellular vesicle conjugates and uses thereof

文档序号：1926227 发布日期：2021-12-03 浏览：22次中文

阅读说明：本技术 胞外囊泡缀合物及其用途 (Extracellular vesicle conjugates and uses thereof ) 是由拉塞尔·E·麦克康纳尔斯里拉姆·萨提亚那拉雅南于 2020-03-20 设计创作，主要内容包括：本公开涉及包含经由马来酰亚胺部分共价连接至所述胞外囊泡的生物活性分子的胞外囊泡(例如,外泌体),所述胞外囊泡可以用作预防或治疗癌症和其他疾病的药剂。本文还提供了用于产生所述胞外囊泡的方法和用于使用所述胞外囊泡来治疗疾病或障碍的方法。(The present disclosure relates to extracellular vesicles (e.g., exosomes) comprising a biologically active molecule covalently attached to the extracellular vesicles via a maleimide moiety, which can be used as agents for preventing or treating cancer and other diseases. Also provided herein are methods for producing the extracellular vesicles and methods for using the extracellular vesicles to treat diseases or disorders.)

1. An Extracellular Vesicle (EV) comprising a biologically active molecule covalently linked to the EV via a maleimide moiety.

2. The extracellular vesicle of claim 1, wherein the maleimide moiety is of formula (I):

wherein

R¹Selected from the group consisting of: -C_1-10Alkylene-, -C_3-8Carbocycle-, -O- (C)_1-8Alkylene) -, -arylene-, -C_1-10Alkylene-arylene-, -arylene-C_1-10Alkylene-, -C_1-10Alkylene- (C)_3-8Carbocycle) -, - (C)_3-8Carbocyclic ring) -C_1-10Alkylene-, -C_3-8Heterocycle-, -C_1-10Alkylene- (C)_3-8Heterocycle) -, - (C)_3-8Heterocycle) -C_1-10Alkylene-, - (CH)₂CH₂O)_r-and- (CH)₂CH₂O)_r-CH₂-；

r is an integer from 1 to 10;

represents the covalent attachment site of the maleimide moiety to the EV; and the number of the first and second electrodes,

the wavy line represents the attachment site of the maleimide moiety to the biologically active molecule.

3. The extracellular vesicle of claim 2, wherein R¹Is- (CH)₂)_s-, where s is 4, 5 or 6.

4. The extracellular vesicle of claim 2 or 3, wherein the maleimide moiety is of formula (II), wherein R¹Is- (CH)₂)₅-：

5. The extracellular vesicle of claim 2, wherein the maleimide moiety is of formula (III), wherein R¹Is- (CH)₂CH₂O)_r-CH₂-, where r is 2:

6. the extracellular vesicle of any one of claims 1-5, wherein the maleimide moiety is covalently attached to a functional group present on the EV, wherein the functional group is a sulfhydryl group.

7. The extracellular vesicle of claim 6, wherein the sulfhydryl group is on a protein of the EV surface.

8. The extracellular vesicle of any one of claims 1-7, wherein the maleimide moiety is linked to the biologically active molecule via a linker.

9. The extracellular vesicle of claim 8, wherein the linker comprises a cleavable linker.

10. The extracellular vesicle of claim 9, wherein the cleavable linker is cleaved by a protease.

11. The extracellular vesicle of claim 10, wherein the protease is a cathepsin.

12. The extracellular vesicle of claim 8, wherein the linker is a reduction-sensitive linker or an acid-labile linker.

13. The extracellular vesicle of any one of claims 8-12, wherein the linker is of formula (IV):

-A_a-Y_y- (IV)，

wherein each-A-is independently an amino acid unit, a is independently an integer from 1 to 12; -Y-is a spacer unit and Y is 0, 1 or 2.

14. The extracellular vesicle of claim 13, wherein-a_a-is a dipeptide, tripeptide, tetrapeptide, pentapeptide or hexapeptide.

15. The extracellular vesicles of claim 14, wherein a is 2, and-a_a-selected from the group consisting of: valine-alanine, valine-citrulline, phenylalanine-lysine, N-methylvaline-citrulline, cyclohexylalanine-lysine and β -alanine-lysine.

16. The extracellular vesicle of claim 15, wherein the-a is_a-is valine-alanine or valine-citrulline.

17. The extracellular vesicle of any one of claims 13-16, wherein y is 1.

18. The extracellular vesicle of any one of claims 13-17, wherein-Y-is a self-immolative spacer.

19. The extracellular vesicle of claim 18, wherein-Y_y-has formula (V):

wherein each R²Independently is C_1-8Alkyl, -O- (C)_1-8Alkyl), halogen, nitro or cyano; and m is an integer of 0 to 4.

20. The extracellular vesicle of claim 19, wherein m is 0, 1, or 2.

21. The extracellular vesicle of claim 20, wherein m is 0.

22. The extracellular vesicle of any one of claims 8-21, wherein the cleavable linker is valine-alanine-p-aminobenzyl carbamate or valine-citrulline-p-aminobenzyl carbamate.

23. The extracellular vesicle of any one of claims 13-17, wherein-Y-is a non-self-immolative spacer.

24. The extracellular vesicle of claim 23, wherein the non-self-immolative spacer is-Gly-or-Gly-.

25. The extracellular vesicle of claim 8, wherein the linker is an acid-labile linker.

26. The extracellular vesicle of claim 25, wherein the acid-labile linker comprises a cis-aconitic acid linker, a hydrazide linker, a thiocarbamoyl linker, or any combination thereof.

27. The extracellular vesicle of claim 25 or 26, wherein the acid-labile linker comprises a spacer unit linking the biologically active molecule to the acid-labile linker.

28. The extracellular vesicle of claim 27, wherein the spacer unit has formula (V):

Wherein each R²Independently is C_1-8Alkyl, -O- (C)_1-8Alkyl), halogen, nitro or cyano; and m is an integer of 0 to 4.

29. The extracellular vesicle of claim 8, wherein the linker is a non-cleavable linker.

30. The extracellular vesicle of claim 29, wherein the non-cleavable linker comprises tetraethylene glycol (TEG), polyethylene glycol (PEG), succinimide, or any combination thereof.

31. The extracellular vesicle of claim 29 or 30, wherein the non-cleavable linker comprises a spacer unit linking the biologically active molecule to the non-cleavable linker.

32. The extracellular vesicle of claim 31, wherein the spacer unit has formula (V):

wherein each R²Independently is C_1-8Alkyl, -O- (C)_1-8Alkyl), halogen, nitro or cyano; and m is an integer of 0 to 4.

33. An extracellular vesicle comprising a biologically active molecule and a cleavable linker, wherein the cleavable linker connects the EV to the biologically active molecule.

34. The extracellular vesicle of claim 33, wherein the cleavable linker comprises valine-alanine-p-aminobenzyl carbamate or valine-citrulline-p-aminobenzyl carbamate.

35. The extracellular vesicle of claim 33 or 34, further comprising a maleimide moiety linking the EV to the cleavable linker via a functional group present on the EV.

36. The extracellular vesicle of claim 35, wherein the maleimide moiety is of formula (I):

wherein

r is an integer from 1 to 10; and is

Represents the covalent attachment site of the maleimide moiety to the EV; and the number of the first and second electrodes,

the wavy line represents the attachment site of the maleimide moiety to the biologically active molecule.

37. The extracellular vesicle of claim 36, wherein R¹Is- (CH)₂)_s-, where s is 4, 5 or 6.

38. The extracellular vesicle of claim 36 or 37, wherein the maleimide moiety is of formula (II), wherein R is¹Is- (CH)₂)₅-：

39. The extracellular vesicle of claim 36, wherein the maleimide moiety is of formula (III), wherein R ¹Is- (CH)₂CH₂O)_r-CH₂-, where r is 2:

40. the extracellular vesicle of any one of claims 34-39, wherein the maleimide moiety is covalently attached to a functional group present on the EV.

41. The extracellular vesicle of claim 40, wherein the functional group is on a glycan on the EV.

42. The extracellular vesicle of claim 40, wherein the functional group is a thiol group.

43. The extracellular vesicle of claim 41 or 42, wherein the functional group is on a protein of the EV surface.

44. The extracellular vesicle of claim 43, wherein the protein is a scaffold moiety.

45. The extracellular vesicle of claim 44, wherein the protein is a PTGFRN polypeptide, a BSG polypeptide, an IGSF2 polypeptide, an IGSF3 polypeptide, an IGSF8 polypeptide, an ITGB1 polypeptide, an ITGA4 polypeptide, a SLC3A2 polypeptide, an ATP translocator polypeptide, or a fragment thereof.

46. An extracellular vesicle comprising a maleimide moiety, a cleavable linker and a bioactive molecule, wherein the maleimide moiety connects the EV to the cleavable linker and the cleavable linker connects the maleimide moiety to the bioactive molecule.

47. The extracellular vesicle of any one of claims 1-46, wherein the biologically active molecule is a polypeptide, a peptide, a polynucleotide (DNA and/or RNA), a compound, or any combination thereof.

48. The extracellular vesicle of claim 47, wherein the biologically active molecule is a compound.

49. The extracellular vesicle of claim 48, wherein the compound is a small molecule.

50. The extracellular vesicle of claim 49, wherein the small molecule is a proteolytic targeting chimera (PROTAC).

51. The extracellular vesicle of claim 47, wherein the biologically active molecule is a nucleotide, wherein the nucleotide is an interferon gene stimulating protein (STING) agonist.

52. The extracellular vesicle of claim 51, wherein the STING agonist comprises a cyclic dinucleotide STING agonist or a non-cyclic dinucleotide STING agonist.

53. The extracellular vesicle of claim 52, comprising a (maleimide moiety) - (cleavable linker) - (bioactive molecule) having formula (VI) or (VII):

(VII) or a pharmaceutically acceptable salt thereof.

54. The extracellular vesicle of claim 52, comprising a (maleimide moiety) - (cleavable linker) - (bioactive molecule) having formula (VIII), (IX), (X) or (XI):

Or a pharmaceutically acceptable salt thereof.

55. The extracellular vesicle of any one of claims 1-5, 8-32, and 35-45, wherein the EV is modified to expose a functional group on a surface covalently attached to the maleimide moiety.

56. The extracellular vesicle of claim 55, wherein the functional group is a sulfhydryl group.

57. The extracellular vesicle of claim 56, wherein the functional group is exposed by treating the EV with a reducing agent.

58. The extracellular vesicle of claim 57, wherein the reducing agent comprises TCEP (tris (2-carboxyethyl) phosphine), DTT (dithiothreitol), BME (2-mercaptoethanol), a thiolating agent, or any combination thereof.

59. The extracellular vesicle of claim 58, wherein the thiolating agent comprises Traut reagent (2-iminothiol).

60. The extracellular vesicle of any one of claims 1-59, wherein the EV is an exosome.

61. A pharmaceutical composition comprising the extracellular vesicle of any one of claims 1-60 and a pharmaceutically acceptable carrier.

62. A method of conjugating a bioactive molecule to an EV, the method comprising attaching a maleimide moiety to the EV.

63. The method of claim 62, wherein the coupling includes treating the EV with a reducing agent.

64. The method of claim 63, wherein the reducing agent comprises TCEP (tris (2-carboxyethyl) phosphine), DTT (dithiothreitol), BME (2-mercaptoethanol), a thiolating agent, or any combination thereof.

65. The method of claim 64, wherein the thiolating agent comprises Traut reagent (2-iminothiol).

66. The method of any one of claims 62 to 65, wherein the ligating further comprises contacting the reduced EV with the maleimide moiety.

67. The method of claim 66, wherein the maleimide moiety is attached to a biologically active molecule prior to attachment to the EV.

68. The method of claim 67, wherein the maleimide moiety is further attached to a linker to attach the maleimide moiety to the biologically active molecule.

69. A kit comprising the EV of any one of claims 1-60 and instructions for use.

70. A kit comprising reagents for conjugating a biologically active molecule to an EV and instructions for performing the conjugation to produce the EV according to any one of claims 1 to 60.

71. A method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering the EV of any one of claims 1-60 to the subject.

72. The method of claim 71, wherein the disease or disorder is cancer, an inflammatory disease, a neurodegenerative disease, a central nervous disease, or a metabolic disease.

73. The method of claim 71 or 72, wherein the EV is administered intravenously, intraperitoneally, nasally, orally, intramuscularly, subcutaneously, parenterally, intratumorally, intrathecally, or intraocularly.

74. An Extracellular Vesicle (EV) comprising at least one biologically active molecule covalently linked to a scaffold moiety via a maleimide moiety.

75. The extracellular vesicle of claim 74, wherein the maleimide moiety is a bifunctional molecule.

76. The extracellular vesicle of claim 74 or 75, wherein the maleimide moiety comprises at least one linker or spacer.

77. The extracellular vesicle of claim 76, wherein the linker is a cleavable linker.

78. The extracellular vesicle of any one of claims 74-77, wherein the scaffold moiety is a scaffold protein or a scaffold lipid.

79. The extracellular vesicle of claim 78, wherein the scaffold protein is a scaffold X protein.

80. The extracellular vesicle of claim 79, wherein the scaffold X protein is a PTGFRN polypeptide, a BSG polypeptide, an IGSF2 polypeptide, an IGSF3 polypeptide, an IGSF8 polypeptide, an ITGB1 polypeptide, an ITGA4 polypeptide, a SLC3A2 polypeptide, an ATP transporter polypeptide, or a fragment thereof.

81. The extracellular vesicle of any one of claims 74-80, wherein the biologically active molecule comprises a vaccine antigen, a vaccine adjuvant, or any combination thereof.

82. The extracellular vesicle of any one of claims 74-80, wherein the biologically active molecule comprises STING, ASO, a synthetic antineoplastic agent (e.g., MMAE), a cytokine release inhibitor (e.g., MCC950), a mTOR inhibitor (e.g., rapamycin), an autotaxin inhibitor (e.g., PAT409), a LPA1 antagonist (e.g., AM152), or any combination thereof.

83. The extracellular vesicle of any one of claims 74-82, wherein the extracellular vesicle further comprises a targeting moiety, a tropism moiety, an anti-phagocytic signal, or any combination thereof.

84. The extracellular vesicle of claim 83, wherein the targeting moiety, tropism moiety, anti-phagocytic signal, or combination thereof is linked to the extracellular vesicle via a maleimide moiety.

Technical Field

The present invention provides Extracellular Vesicles (EVs) (e.g., exosomes) comprising at least one bioactive molecule covalently attached via a maleimide moiety to the extracellular vesicles (e.g., exosomes), which can be used as agents for preventing or treating cancer and other diseases.

Background

Many biologically active compounds have therapeutically useful biological activities. However, these compounds often exhibit toxicity in non-target organs. One way to limit non-target tissue exposure is to chemically conjugate small molecules to affinity-based agents, such as antibodies, which can direct therapeutic compounds to specific cell types (Dosio, f. et al, toxins (basel)3(7):848-883(2011)), but this approach is limited by the number of molecules of the compound of interest that can be attached to the antibody (typically 2-6 molecules per antibody) and the availability/presence of antibodies that specifically bind to the targeted relevant diseased/effector cells but not to the non-target cells. These two problems limit the use of antibody-drug conjugates (ADCs) by reducing potency and increasing systemic toxicity, respectively. Thus, there is a need for delivery systems with higher payloads than ADCs that can selectively target specific tissues or organs while limiting the overall systemic exposure of the therapeutic compound.

EV (e.g., exosomes) are important mediators of cell-cell communication. They are also important biomarkers for the diagnosis and prognosis of many diseases, such as cancer. As a drug delivery vehicle, EV (e.g., exosomes) are a novel therapeutic modality in many therapeutic areas, with many advantages over traditional drug delivery methods (e.g., peptide immunization, DNA vaccines). However, despite their advantages, many EVs (e.g., exosomes) have limited clinical efficacy. For example, dendritic cell-Derived Exosomes (DEX) were studied as maintenance immunotherapy in phase II clinical trials following first-line chemotherapy in inoperable non-small cell lung cancer (NSCLC) patients. However, the trial was terminated because the primary endpoint was not reached (at least 50% of patients had a Progression Free Survival (PFS) of 4 months after chemotherapy was discontinued). Besse, B. et al, Onco immunology 5(4) e1071008 (2015).

Therefore, new and more efficient engineered EVs (e.g., exosomes) are necessary to better enable therapeutic use of EV-based technologies and other applications.

Disclosure of Invention

The present disclosure provides an extracellular vesicle (e.g., exosome) comprising a bioactive molecule covalently attached to an EV (e.g., exosome) via a maleimide moiety. In some aspects, the maleimide moiety has formula (I):

wherein

r is an integer from 1 to 10;

represents a covalent attachment site of the maleimide moiety to the EV (e.g., exosome); and the number of the first and second electrodes,

the wavy line represents the attachment site of the maleimide moiety to the biologically active molecule.

In some aspects, R¹Is- (CH)₂)_s-, where s is 4, 5 or 6. In some aspects, the maleimide moiety has formula (II), wherein R¹Is- (CH)₂)₅-：

In some aspects, the maleimide moiety has formula (III), wherein R ¹Is- (CH)₂CH₂O)_r-CH₂-, where r is 2:

in some aspects, the maleimide moiety is covalently attached to a functional group present on the EV (e.g., exosome), wherein the functional group is a thiol group. In some aspects, the sulfhydryl group is on a protein on the surface of the EV (e.g., exosome). In some aspects, the maleimide moiety is attached to the biologically active molecule via a linker. In some aspects, the linker comprises a cleavable linker. In some aspects, the cleavable linker is cleaved by a protease. In some aspects, the protease is a cathepsin. In some aspects, the linker is a reduction-sensitive linker or an acid-labile linker.

In some aspects, the linker has formula (IV):

-A_a-Y_y- (IV)，

wherein each-A-is independently an amino acid unit, a is independently an integer from 1 to 12; -Y-is a spacer unit and Y is 0, 1 or 2.

In some aspects, -A_a-is a dipeptide, tripeptide, tetrapeptide, pentapeptide or hexapeptide. In some aspects, a is 2, and-A_a-selected from the group consisting of: valine-alanine, valine-citrulline, phenylalanine-lysine, N-methylvaline-citrulline, cyclohexylalanine-lysine and β -alanine-lysine. In some aspects, -A _a-is valine-alanine or valine-citrulline. In some aspects, y is 1.

In some aspects, -Y-is a self-immolative spacer. In some aspects, -Y_y-has formula (V):

wherein each R²Independently is C_1-8Alkyl, -O- (C)_1-8Alkyl), halogen, nitro or cyano; and m is an integer of 0 to 4.

In some aspects, m is 0, 1, or 2. In some aspects, m is 0. In some aspects, the cleavable linker is valine-alanine-p-aminobenzyl carbamate or valine-citrulline-p-aminobenzyl carbamate. In some aspects, -Y-is a non-self-immolative spacer. In some aspects, the non-self-immolative spacer is-Gly-or-Gly-.

In some aspects, the linker is an "acid labile linker". In some aspects, the acid-labile linker comprises a cis-aconitic acid linker, a hydrazide linker, a thiocarbamoyl linker, or any combination thereof. In some aspects, the acid-labile linker comprises a spacer unit that links the biologically active molecule to the acid-labile linker.

In some aspects, the spacer unit has formula (V):

wherein each R²Independently is C_1-8Alkyl, -O- (C) _1-8Alkyl), halogen, nitro or cyano; and m is an integer of 0 to 4.

In some aspects, the linker is a non-cleavable linker. In some aspects, the non-cleavable linker comprises tetraethylene glycol (TEG), polyethylene glycol (PEG), succinimide, or any combination thereof. In some aspects, the non-cleavable linker comprises a spacer unit that links the biologically active molecule to the non-cleavable linker. In some aspects, the spacer unit has formula (V):

wherein each R²Independently is C_1-8Alkyl, -O- (C)_1-8Alkyl), halogen, nitro or cyano; and m is an integer of 0 to 4.

The present disclosure also provides an EV (e.g., exosome) comprising a bioactive molecule and a cleavable linker, wherein the cleavable linker connects the EV (e.g., exosome) to the bioactive molecule, and the cleavable linker comprises valine-alanine-p-aminobenzyl carbamate or valine-citrulline-p-aminobenzyl carbamate. In some aspects, the EV (e.g., exosome) further comprises a maleimide moiety that links the EV (e.g., exosome) to the cleavable linker via a functional group present on the EV (e.g., exosome).

In some aspects, the maleimide moiety has formula (I):

wherein

R¹Selected from the group consisting ofGroup (b): -C_1-10Alkylene-, -C_3-8Carbocycle-, -O- (C)_1-8Alkylene) -, -arylene-, -C_1-10Alkylene-arylene-, -arylene-C_1-10Alkylene-, -C_1-10Alkylene- (C)_3-8Carbocycle) -, - (C)_3-8Carbocyclic ring) -C_1-10Alkylene-, -C_3-8Heterocycle-, -C_1-10Alkylene- (C)_3-8Heterocycle) -, - (C)_3-8Heterocycle) -C_1-10Alkylene-, - (CH)₂CH₂O)_r-and- (CH)₂CH₂O)_r-CH₂-；

r is an integer from 1 to 10; and is

Represents a covalent attachment site of the maleimide moiety to the EV (e.g., exosome); and the number of the first and second electrodes,

the wavy line represents the attachment site of the maleimide moiety to the biologically active molecule.

In some aspects, R¹Is- (CH2) s-, wherein s is 4, 5 or 6. In some aspects, the maleimide moiety has formula (II), wherein R¹Is- (CH)₂)₅-：

In some aspects, the maleimide moiety has formula (III), wherein R¹Is- (CH)₂CH₂O)_r-CH₂-, where r is 2:

in some aspects, the maleimide moiety is covalently attached to a functional group present on the EV (e.g., exosome). In some aspects, the functional group is on a glycan on the EV (e.g., exosome). In some aspects, the functional group is a thiol (thiol). In some aspects, the functional group is on a protein on the surface of the EV (e.g., exosome). In some aspects, the protein is a scaffold moiety. In some aspects, the protein is a PTGFRN polypeptide, a BSG polypeptide, an IGSF2 polypeptide, an IGSF3 polypeptide, an IGSF8 polypeptide, an ITGB1 polypeptide, an ITGA4 polypeptide, a SLC3a2 polypeptide, an ATP transporter polypeptide, or a fragment thereof.

The present disclosure also provides an EV (e.g., exosome) comprising a maleimide moiety, a cleavable linker and a bioactive molecule, wherein the maleimide moiety connects the EV (e.g., exosome) to the cleavable linker and the cleavable linker connects the maleimide moiety to the bioactive molecule.

In some aspects, the bioactive molecule is a polypeptide, a peptide, a polynucleotide (DNA and/or RNA), a chemical compound, or any combination thereof. In some aspects, the biologically active molecule is a chemical compound. In some aspects, the chemical compound is a small molecule. In some aspects, the small molecule is a proteolytic targeting chimera (PROTAC).

In some aspects, the biologically active molecule is a nucleotide, wherein the nucleotide is an interferon gene stimulating protein (STING) agonist. In some aspects, the STING agonist comprises a cyclic-dinucleotide STING agonist or a non-cyclic-dinucleotide STING agonist.

In some aspects, the EV comprises a (maleimide moiety) - (cleavable linker) - (bioactive molecule) having formula (VI) or (VII):

or a pharmaceutically acceptable salt thereof.

In some aspects, the EV comprises a (maleimide moiety) - (cleavable linker) - (biologically active molecule) having formula (VIII), (IX), (X), or (XI):

or a pharmaceutically acceptable salt thereof.

In some aspects, the EV (e.g., exosome) is modified to expose a functional group on the surface to which the maleimide moiety is covalently attached. In some aspects, the functional group is a thiol group. In some aspects, the functional group is exposed by treating the EV (e.g., exosome) with a reducing agent. In some aspects, the reducing agent comprises TCEP (tris (2-carboxyethyl) phosphine), DTT (dithiothreitol), BME (2-mercaptoethanol), a thiolating agent, or any combination thereof. In some aspects, the thiolating agent includes Traut's reagent (2-iminothiolane).

In various aspects, the EVs of the present disclosure include exosomes.

The present disclosure also provides a pharmaceutical composition comprising an EV (e.g., exosome) disclosed herein and a pharmaceutically acceptable carrier.

The present disclosure also provides a method of conjugating a bioactive molecule to an EV (e.g., an exosome), the method comprising attaching a maleimide moiety to the EV (e.g., exosome). In some aspects, the linking comprises treating the EV (e.g., exosomes) with a reducing agent. In some aspects, the reducing agent is a composition comprising TCEP (tris (2-carboxyethyl) phosphine), DTT (dithiothreitol), BME (2-mercaptoethanol), a thiolating agent, or any combination thereof. In some aspects, the thiolating agent includes Traut's reagent (2-iminothiolane). In some aspects, the linking further comprises contacting the reduced EV (e.g., exosome) with the maleimide moiety. In some aspects, the maleimide moiety is chemically attached to a biologically active molecule prior to attachment to the EV (e.g., exosome). In some aspects, the maleimide moiety is chemically linked to a linker to link the maleimide moiety to the biologically active molecule.

The present disclosure also provides a kit comprising an EV (e.g., exosome) disclosed herein and instructions for use. Also provided is a kit comprising an agent for conjugating a biologically active molecule to an EV (e.g., an exosome) and instructions for performing the conjugation to prepare an EV (e.g., an exosome) of the present disclosure.

The present disclosure also provides a method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering an EV (e.g., exosome) of the present disclosure to the subject. In some aspects, the disease or disorder is cancer, an inflammatory disorder, a neurodegenerative disorder, a central nervous disease, or a metabolic disease. In some aspects, the EV (e.g., exosome) is administered intravenously, intraperitoneally, nasally, orally, intramuscularly, subcutaneously, parenterally, intrathecally, intraocularly, or intratumorally.

In some aspects, the present disclosure provides an Extracellular Vesicle (EV) comprising at least one biologically active molecule covalently attached to a scaffold moiety via a maleimide moiety. In some aspects, the maleimide moiety is a bifunctional molecule. In some aspects, the maleimide moiety comprises at least one linker or spacer. In some aspects, the linker is a cleavable linker. In some aspects, the scaffold moiety is a scaffold protein or a scaffold lipid. In some aspects, the scaffold protein is a scaffold X protein. In some aspects, the scaffold X protein is a PTGFRN polypeptide, a BSG polypeptide, an IGSF2 polypeptide, an IGSF3 polypeptide, an IGSF8 polypeptide, an ITGB1 polypeptide, an ITGA4 polypeptide, a SLC3a2 polypeptide, an ATP transporter polypeptide, or a fragment thereof. In some aspects, the bioactive molecule comprises a vaccine antigen, a vaccine adjuvant, or any combination thereof. In some aspects, the bioactive molecule comprises STING, ASO, a synthetic antineoplastic agent (e.g., MMAE), a cytokine release inhibitor (e.g., MCC950), a mTOR inhibitor (e.g., rapamycin), an autotaxin inhibitor (e.g., PAT409), a LPA1 antagonist (e.g., AM152), or any combination thereof. In some aspects, the extracellular vesicle further comprises a targeting moiety, a tropism moiety, an anti-phagocytic signal, or any combination thereof. In some aspects, the targeting moiety, tropism moiety, anti-phagocytic signal, or combination thereof is linked to the extracellular vesicle via a maleimide moiety.

Drawings

Fig. 1A is a schematic diagram showing how bioactive molecules (BAMs) are chemically linked to EVs (e.g., exosomes) using maleimide chemistry, for example via a scaffold moiety (e.g., scaffold X protein or fragment thereof or lipid) as described herein. The linkers depicted in the figures are optional and, when present, can include linkers (e.g., cleavable linkers) or combinations thereof.

Figure 1B shows an example of STING agonist compounds that can be linked to EV: CP227 (Val-Ala attached to the maleimide moiety), CP229 (Val-Cit attached to the maleimide moiety), CP238 (Val-Ala attached to cholesterol), CP246 (Val-Ala attached to the maleimide moiety), CP240 (no linker), CP249 (Val-Ala attached to the maleimide moiety), CP250 (Val-Ala attached to the maleimide moiety), CP260 (Val-Cit attached to the maleimide moiety), and CP261 (Val-Cit attached to the maleimide moiety).

FIG. 2 shows the results of a PBMC assay to evaluate the STING agonism (IFN-. beta.) of thiol or amine reactive compounds. Free STING agonist compounds (filled circles) and STING agonist compounds loaded on exosomes (open circles) were tested for activity. Exo CP227 is CP227 conjugated to EV via Val-Ala linked to a maleimide moiety; Exo-CP229 is CP229 conjugated to EV via Val-Cit linked to a maleimide moiety; Exo-CP232 is CP232 conjugated to EV without any linker; Exo-CP246 is CP246 conjugated to EV via Val-Cit linked to succinimide; and Exo-CP250 is CP250 conjugated to EV via Val-Cit linked to a maleimide moiety. 500,000 PBMCs/well were incubated with exosomes overnight. The release of interferon beta (IFN β) into the cell culture supernatant was measured using ELISA.

Figures 3A-3C show the results of PBMC assays comparing thiol reactivity and lipid-associated chemistry against loading STING agonists (central panel). FIG. 3A shows IFN beta release and EC for CP227 and ExoCP227₅₀In comparison, the latter is conjugated to CP227 of the EV via Val-Ala linked to a maleimide moiety. FIG. 3B shows IFN beta release and EC for CP229 and ExoCP229₅₀In comparison, the latter is conjugated to E via Val-Cit linked to a maleimide moietyV CP 229. FIG. 3C shows IFN beta release and EC for CP238 and ExoCP238₅₀In comparison, the latter is conjugated to CP238 of EV via Val-Ala linked to cholesterol.

Figure 4A shows comparative data on IFN β release in PBMC assay between ADUS100 and ExoADUS100, the latter being ADUS100 encapsulated in EV (in cavity). Figure 4B shows comparative data on IFN β release in PBMC assay between CL656 and ExoCL656, which is CL656 encapsulated in EV (in cavity). FIG. 4C shows the EC50 for the various STING agonists and Exo-STING agonists described in FIGS. 3A-3C and 4A-4B.

FIG. 5A shows the loading efficiency of an EV: the exoCP227, the exoCP229, the exoCP250, the exoCP238, the exoCP232, and the exoCP 246. The structure of the EV is described above. The number of STING agonists loaded on each EV in both experiments is shown. FIG. 5B shows EC for various STING agonists and EVs ₅₀And (3) comparison: CP227, exo-CP227, CP229, exo-CP229, CP238, exo-CP238, ADUS100, exoADUS100, CL656, exoCL656, and exoCP 250.

FIG. 6 shows the structures of monomethyl auristatin E (MMAE) and maleimide-Val-Cit-PABC-MMAE (vc-MMAE).

Figure 7 shows MMAE cytotoxicity assessed against RAW264.7(RAW) cells (human macrophage cell line). The dose-response effect of MMAE on RAW cell growth is shown in the micrograph (top), cell growth measurements (bottom left), and confluence (bottom right).

FIG. 8A provides pooled data comparing the efficacy of MMAE (MMAE) in free form or MMAE with a maleimide-Val-Cit-PABC linker (vc-MMAE).

Figure 8B provides pooled data measuring the efficacy of MMAE after exosome clearance after exosome incubation with free MMAE. The exosomes were washed with guanidine hydrochloride at a concentration between 0.1M and 2M.

FIG. 8C provides pooled data measuring the efficacy of MMAE after exosome clearance after exosome incubation with Val-Cit-MMAE. The exosomes were washed with guanidine hydrochloride at a concentration between 0.1M and 2M.

FIG. 8D provides pooled data measuring the efficacy of Val-Cit-MMAE or free MMAE after incubation of exosomes with the MMAE under reducing or non-reducing conditions. Exosomes were incubated with Val-Cit-MMAE or MMAE in the presence or absence of 5mM TCEP.

FIG. 9A shows the effect of reducing conditions (0mM TCEP to 50mM TCEP), loading concentrations of compounds (10 μ M to 100 μ M vc-MMAE) and the presence or absence of guanidine hydrochloride (0M or 1M) on the potency of Val-Cit-MMAE loaded exosomes. FIG. 9B shows the effect of reducing conditions (0mM TCEP to 50mM TCEP), loading concentrations of compounds (100. mu.M or 300. mu.M vc-MMAE) and the presence or absence of guanidine hydrochloride (0M or 1M) on the potency of Val-Cit-MMAE loaded exosomes.

Fig. 10A shows a schematic of PROTAC (proteolytic targeting chimera).

Fig. 10B shows a schematic diagram of the mechanism of action of PROTAC.

Fig. 10C shows a formula corresponding to PROTAC comprising a VHL (E3 ligase) binding ligand moiety, a linker and a TBK1(TANK binding kinase 1) targeting ligand. This formula shows potential sites (represented as stars) on the VHL (E3 ligase) binding ligand moiety that are easily derivatized with maleimide linkers to chemically attach the PROTAC to extracellular vesicles (e.g., exosomes).

FIG. 11 is a schematic of the mechanism of action of CLIPTAT.

FIG. 12 shows the chemical structures of AM152 (cyclopropanecarboxylic acid, 1- [4'- [ 3-methyl-4- [ [ [ (1R) -1-phenylethoxy ] carbonyl ] amino ] -5-isoxazolyl ] [1,1' -biphenyl ] -4-yl ] -) and AM095(1,1 '-biphenyl ] -4-acetic acid, 4' - [ 3-methyl-4- [ [ [ (1R) -1-phenylethoxy ] carbonyl ] amino ] -5-isoxazolyl [ ] -). Arrows labeled 1 and 2 indicate positions (formate and carbamate) suitable for derivatization to introduce maleimide reactive groups. The corresponding site indicated in AM152 is also present in AM 095.

Fig. 13 is a schematic showing LPA1 antagonist (AM152) conjugated to exosomes to generate a population of exosomes containing multiple LPA1 antagonist molecules on the surface.

Figure 14 shows an example of how a maleimide reactive group can be added to AM152 via its carboxylic acid group. This example shows a maleimide group as part of a reactive complex comprising an Ala-Val cleavable linker and a C5 spacer interposed between the maleimide group and the carboxylic acid-reactive chloromethyl phenyl group.

Figure 15 shows two exemplary reagents that can be used to derivatize AM 152. The above reagents contained (i) a chloromethylphenyl group which could react with the carboxylic acid group of AM152 and (ii) a maleimide group. A cleavable Cit-Val dipeptide and a C5 spacer are inserted between (i) and (ii). The following reagents comprise (i) a chloromethylphenyl group which can react with the carboxylic acid group of AM152 and (ii) a maleimide group, and a cleavable Ala-Val dipeptide and a C5 spacer are inserted between (i) and (ii).

FIG. 16 shows the products resulting from cleavage of the Cit-Val or Ala-Val dipeptide in the conjugate product (e.g., by cathepsin B). This product AM152 anilino ester can be further processed by endogenous esterases to yield the free acid AM152 product.

Figures 17 and 18 show several AM152 derivatives containing free maleimide groups and different spacer combinations.

Fig. 19 shows that following protection of the carboxylic acid groups, the same reagents used to derivatize the carboxylic acid groups can be used to derivatize AM152 at the carbamate groups. The resulting product is then deprotected to release the carboxylic acid group.

Fig. 20 shows a display example in which a complex with a maleimide group is chemically linked to a carbamate group of AM152 via a linker. Suitable linkers include any of the linkers disclosed in this specification.

Figure 21 shows that AM152 can be chemically attached to a derivatized scaffold moiety, rather than derivatized and then attached to a scaffold moiety via a reactive maleimide group.

Figure 22 shows the structures of (i) MCC950, (ii) bifunctional reagents that can be used to derivatize MCC950 to introduce maleimide-reactive groups, and (iii) MCC950 derivatives containing maleimide-reactive groups. The phenyl group (×) of the bifunctional reagent may react with the carbamate group (×) of MCC950 to produce the described MCC950 derivatives.

Detailed Description

The present disclosure relates to Extracellular Vesicles (EVs) (e.g., exosomes) comprising at least one biologically active molecule covalently attached to an EV (e.g., exosome) via a maleimide moiety and their uses. EVs (e.g., exosomes) comprising bioactive molecules linked via a maleimide moiety show superior properties compared to conventional moieties (e.g., cholesterol or succinimides). Non-limiting examples of various aspects are shown in this disclosure.

Before the present disclosure is described in greater detail, it is to be understood that this invention is not limited to particular compositions or process steps described, as such may, of course, vary. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the present invention. Any recited method may be performed in the order of the recited events or in any other order that is logically possible.

The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Accordingly, the terms defined directly below are defined in more detail by reference to the specification as a whole.

I. Definition of

In order to make the present description easier to understand, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

It is noted that the term "an" entity refers to one or more of that entity; for example, "nucleotide sequence" is understood to mean one or more nucleotide sequences. Thus, the terms "a" or "an", "one or more" and "at least one" may be used interchangeably herein. It is further noted that the claims may be formulated to exclude any optional elements. Accordingly, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," etc., or use of a "negative" limitation in connection with the recitation of claim elements.

Further, as used herein, "and/or" should be taken as specifically disclosing the presence of each of the two specified features or components, with or without the other. Thus, the term "and/or" as used in phrases such as "a and/or B" is intended to include "a and B," "a or B," "a" (alone), and "B" (alone). Also, the use of the term "and/or" as in phrases such as "A, B and/or C" is intended to include each of the following: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

It should be understood that wherever the term "comprising" is used herein to describe an aspect, other similar aspects described as "consisting of and/or" consisting essentially of.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates. For example, the circumcise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2 nd edition, 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 3 rd edition, 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised,2000, Oxford University Press provide the skilled artisan with a general Dictionary Of many Of the terms used in this disclosure.

Units, prefixes, and symbols are represented in their International system of units (Systeme International de units) (SI) approved form. Numerical ranges include the numbers defining the range. Where a range of values is recited, it is understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, as well as each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the disclosure. Accordingly, recitation of ranges herein are intended to serve as a shorthand method of referring individually to all values falling within the range, including the endpoints recited. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

Where values are explicitly recited, it is understood that values that are about the same as the recited values in quantity or amount are also within the scope of the disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are disclosed individually, combinations of the elements or groups of elements are also disclosed. Where any element of the present disclosure is disclosed as having a plurality of alternatives, examples of the present disclosure in which each alternative is excluded alone or in any combination with the other alternatives are also hereby disclosed; more than one element of the present disclosure may have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

Nucleotides are referred to by their commonly accepted single letter codes. Nucleotide sequences are written in a 5 'to 3' direction from left to right unless otherwise indicated. Nucleotides are referred to herein by their well-known single letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission. Thus, a represents adenine, C represents cytosine, G represents guanine, T represents thymine, U represents uracil.

Amino acid sequences are written from left to right in the amino to carboxyl direction. Amino acids may be referred to herein by their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission.

The term "about" is used herein to mean approximately, on the left and right, or within. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the upper and lower limits of the numerical values set forth. In general, the term "about" can modify values above and below the stated value by, for example, a variance of 10%, above or below (higher or lower).

The term "administration" and grammatical variations thereof refers to introducing a composition of the present disclosure, such as an EV (e.g., exosome), into a subject via a pharmaceutically acceptable route. The compositions of the present disclosure, such as EVs (e.g., exosomes), are introduced into a subject by any suitable route, including intratumoral, oral, intrapulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intrathecal, periocular, or topical routes. Administration includes self-administration and administration by others. Suitable routes of administration allow the composition or agent to perform its intended function. For example, if the suitable route is intravenous route, the composition is administered by introducing the composition or agent into the vein of the subject.

As used herein, the term "agonist" refers to a molecule that binds to a receptor and activates the receptor to produce a biological response. Receptors can be activated by endogenous or exogenous agonists. Non-limiting examples of endogenous agonists include hormones, neurotransmitters and cyclic dinucleotides. Non-limiting examples of exogenous agonists include drugs, small molecules, and cyclic dinucleotides. Agonists may be full, partial or inverse agonists.

The term "amino acid substitution" refers to the replacement of one amino acid residue present in a parent or reference sequence (e.g., a wild-type sequence) with another amino acid residue. For example, amino acids can be substituted in a parent or reference sequence (e.g., a wild-type polypeptide sequence) via chemical peptide synthesis or by recombinant methods known in the art. Thus, reference to "substitution at position X" means that the amino acid present at position X is substituted with another amino acid residue. In some aspects, the substitution pattern can be described according to pattern AnY, wherein a is the one letter code corresponding to the amino acid naturally or initially present at position n, and Y is the substituting amino acid residue. In other aspects, the substitution patterns can be described in terms of the patterns an (yz), where a is the one letter code corresponding to the amino acid residue that is substituted for the amino acid naturally or initially present at position n, and Y and Z are alternatives for substituting the amino acid residue that can be substituted for a.

As used herein, the term "antagonist" refers to a molecule that blocks or inhibits an agonist-mediated response, rather than a molecule that itself elicits a biological response upon binding to a receptor. Many antagonists achieve their efficacy by competing with endogenous ligands or substrates at structurally defined binding sites on the receptor. Non-limiting examples of antagonists include alpha blockers, beta blockers, and calcium channel blockers. The antagonist may be a competitive, non-competitive antagonist or a non-competitive antagonist.

As used herein, the term "antibody" encompasses immunoglobulins and fragments thereof, whether naturally occurring or partially or wholly synthetically produced. The term also encompasses any protein having a binding domain that is homologous to an immunoglobulin binding domain. "antibodies" also include polypeptides comprising framework regions from immunoglobulin genes or fragments thereof that specifically bind to and recognize antigens. The use of the term antibody is intended to include complete, polyclonal, monoclonal and recombinant antibodies, fragments thereof, and also single chain antibodies, humanized antibodies, murine antibodies, chimeric monoclonal antibodies, mouse-human monoclonal antibodies, mouse-primate monoclonal antibodies, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments (such as, for example, scFv, (scFv) ₂Fab, Fab 'and F (ab')₂、F(ab1)₂Fv, dAb and Fd fragments), diabodies and antibody-related polypeptides. Antibodies include bispecific antibodies and multispecific antibodies so long as they exhibit the desired biological activity or function. In some aspects of the disclosure, the biologically active molecule is an antibody or a molecule comprising an antigen binding fragment thereof.

The terms "antibody-drug conjugate" and "ADC" are used interchangeably to refer to an antibody that is linked (e.g., covalently linked) to one or more therapeutic agents (sometimes referred to herein as agents, drugs, or active pharmaceutical ingredients). In some aspects of the disclosure, the bioactive molecule is an antibody-drug conjugate.

As used herein, the term "about" when applied to one or more desired values refers to a value that is similar to the referenced value. In certain aspects, the term "about" refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the referenced value, unless otherwise indicated or otherwise evident from the context (unless the number exceeds 100% of the possible values).

The term "aryl" refers to a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl. Carbocyclic aromatic groups may be unsubstituted or substituted with one or more groups including, but not limited to-C _1-8Alkyl, -O- (C)_1-8Alkyl), -aryl, -C (O) R ', -OC (O) R ', -C (O) OR ', -C (O) NH₂、-C(O)NHR'、-C(O)N(R')₂-、-NHC(O)R'、-S(O)₂R ', -S (O) R', -OH, -halogen, -N₃、-NH₂、-NH(R')、-N(R')₂and-CN, wherein each R' is independently H, -C_1-8Alkyl or aryl.

The term "arylene" refers to an aryl group having two covalent bonds and may be in the ortho, meta, or para configuration, as shown in the following structure:

wherein the phenyl group may be unsubstituted or substituted with up to four groups including, but not limited to-C_1-8Alkyl, -O- (C)_1-8Alkyl), -aryl, -C (O) R ', -OC (O) R ', -C (O) OR ', -C (O) NH₂、-C(O)NHR'、-C(O)N(R')₂-、-NHC(O)R'、-S(O)₂R ', -S (O) R', -OH, -halogen, -N₃、-NH₂、-NH(R')、-N(R')₂and-CN, wherein each R' is independently H, -C_1-8Alkyl or aryl.

As used herein, the term "bioactive molecule" refers to any molecule that can be linked (e.g., chemically linked via a maleimide moiety) to an EV (e.g., exosome), wherein the molecule can have a therapeutic or prophylactic effect in a subject in need thereof, or for diagnostic purposes. Thus, for example, the term bioactive molecule includes proteins (e.g., antibodies, proteins, polypeptides and derivatives, fragments and variants thereof), lipids and derivatives thereof, carbohydrates (e.g., glycan moieties in glycoproteins), or small molecules. In some aspects, the biologically active molecule is a radioisotope. In some aspects, the bioactive molecule is a detectable moiety, e.g., a radionuclide, a fluorescent molecule, or a contrast agent.

As used herein, the term "C_1-8Alkyl "refers to straight or branched chain saturated hydrocarbons having 1 to 8 carbon atoms. Representative of "C_1-8Alkyl "groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and 2-methylbutyl.

The term "C_1-10Alkylene "means a group of the formula- (CH)₂)_1-10A saturated linear hydrocarbon group of (A-A). C_1-10Examples of alkylene groups include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, and decylene.

The term "C_3-8Carbocycle "refers to a 3-, 4-, 5-, 6-, 7-or 8-membered saturated or unsaturated non-aromatic carbocyclic ring. Representative C_3-8Carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1, 3-cyclohexadienyl, 1, 4-cyclohexadienyl, cycloheptyl, 1, 3-cycloheptadienyl, 1,3, 5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl. C_3-8The carbocyclic group may be unsubstituted or substituted with one or more groups including, but not limited to-C_1-8Alkyl, -O- (C)_1-8Alkyl), aryl, -C (O) R ', -OC (O) R ', -C (O) OR ', -C (O) NH ₂、-C(O)NHR'、-C(O)N(R')₂-、NHC(O)R'、-S(O)₂R ', -S (O) R', -OH, -halogen, -N₃、-NH₂、-NH(R')、-N(R')₂and-CN, wherein each R' is independently H, -C_1-8Alkyl or aryl.

The term "C_3-8Carbocycle "means C as defined above_3-8Carbocyclic groups in which one or more of the carbocyclic hydrogen atoms are substituted by a bond.

The term "C_3-8Heterocycle "means an aromatic or non-aromatic C_3-8A carbocycle wherein one to four of the ring carbon atoms are independently substituted with a heteroatom selected from the group consisting of: o, S and N. C_3-8Representative examples of heterocycles include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolyl, pyrrolyl, thienyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, and tetrazolyl. C_3-8The heterocyclic ring may be unsubstituted or substituted with up to seven groups including, but not limited to-C_1-8Alkyl, -O- (C)_1-8Alkyl), -aryl, -C (O) R ', -OC (O) R ', -C (O) OR ', -C (O) NH₂、-C(O)NHR'、-C(O)N(R')₂、-NHC(O)R'、-S(O)₂R ', -S (O) R', -OH, -halogen, -N₃、-NH₂、-NH(R')、-N(R')₂and-CN, wherein each R' is independently H, -C_1-8Alkyl or aryl.

The term "C_3-8Heterocycle "means C as defined above _3-8A heterocyclic group wherein one of the hydrogen atoms of the heterocyclic group is substituted by a bond. C_3-8The heterocyclic ring may be unsubstituted or substituted with up to six groups including, but not limited to-C_1-8Alkyl, -O- (C)_1-8Alkyl), -aryl, -C (O) R ', -OC (O) R ', -C (O) OR ', -C (O) NH₂、-C(O)NHR'、-C(O)N(R')₂、-NHC(O)R'、-S(O)₂R ', -S (O) R', -OH, -halogen, -N₃、-NH₂、-NH(R')、-N(R')₂and-CN, wherein each R' is independently H, -C_1-8Alkyl or aryl.

A "conservative amino acid substitution" is a substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, such a substitution is considered conservative. In another aspect, the amino acid string can be conservatively substituted with a string that differs in the order and/or composition of the side chain family members but is structurally similar.

As used herein, the term "conserved" refers to nucleotide or amino acid residues of a polynucleotide sequence or polypeptide sequence, respectively, that are unchanged at the same position of two or more sequences being compared. A nucleotide or amino acid that is relatively conserved is one that is conserved in a sequence more related than nucleotides or amino acids present elsewhere in the sequence.

In some aspects, two or more sequences are said to be "fully conserved" or "identical" if they are 100% identical to each other. In some aspects, two or more sequences are said to be "highly conserved" if they are at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to each other. In some aspects, two or more sequences are said to be "conserved" if they are at least about 30% identical, at least about 40% identical, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to each other. Sequence conservation may apply to the full length of the polynucleotide or polypeptide or may apply to portions, regions or features thereof.

As used herein, the term "conventional EV protein" means a protein previously known to be enriched in an EV.

As used herein, the term "conventional exosome protein" means a protein previously known to be enriched in exosomes, including, but not limited to, CD9, CD63, CD81, PDGFR, GPI proteins, lectin LAMP2, and LAMP2B, fragments thereof, or peptides that bind thereto.

As used herein, the term "derivative" refers to an EV (e.g., exosome), a component (e.g., scaffold protein, such as scaffold X and/or scaffold Y, lipid or carbohydrate), or a biologically active molecule (e.g., polypeptide, polynucleotide, lipid, carbohydrate, antibody or fragment thereof, PROTAC, etc.) that has been chemically modified to introduce a reactive maleimide group or a thiol group that is susceptible to reaction with a maleimide group. For example, antibodies modified with bifunctional reagents comprising (i) a group that reacts, for example, with a free amino group, and (ii) a maleimide group that can react with a free thiol group in a scaffold X protein on an EV (e.g., exosome) can produce antibody derivatives comprising a reactive maleimide group. In contrast, scaffold X on EVs (e.g., exosomes) may be modified with bifunctional reagents comprising (i) a group reactive with, for example, a free amino group, and (ii) a maleimide group, which reactive maleimide group may react with a free thiol group in a biologically active molecule (e.g., antibody), to produce a derivative of scaffold X comprising a reactive maleimide group.

The terms "excipient" and "carrier" are used interchangeably to refer to an inert substance added to a pharmaceutical composition to further facilitate administration of the compound.

As used herein, the terms "extracellular vesicle," "EV," and grammatical variations thereof, are used interchangeably to refer to a cell-derived vesicle comprising a membrane that encapsulates an interior space. Extracellular vesicles include all membrane-bound vesicles (e.g., exosomes, nanovesicles) whose diameter is smaller than the diameter of the cell from which they are derived. In some aspects, the extracellular vesicles have diameters in the range of 20nm to 1000nm, and may include various macromolecular payloads within the interior space (i.e., cavity), displayed on the outer surface of the extracellular vesicles, and/or across the membrane. In some aspects, the payload can include an adeno-associated virus (AAV), a nucleic acid (e.g., DNA or RNA, such as an antisense oligonucleotide, siRNA, shRNA, or mRNA), a morpholine, a protein, a carbohydrate, a lipid, a small molecule, an antigen, a vaccine adjuvant, and/or combinations thereof. Additional payloads are described in detail below. In some aspects, an EV (e.g., exosome) may further comprise a targeting moiety, a tropism moiety, or a combination thereof. In some aspects, the term extracellular vesicles or EVs refers to a population of Extracellular Vesicles (EVs).

In certain aspects, the extracellular medium comprises a scaffold moiety. By way of example and not limitation, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with an alkaline solution), vesicles containing vesicles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusing late endosomes to the plasma membrane). The extracellular vesicles may be derived from living or dead organisms, explanted tissues or organs, prokaryotic or eukaryotic cells, and/or cultured cells. In some aspects, the extracellular vesicles are produced by cells expressing one or more transgene products.

The term "exosome" as used herein refers to an extracellular vesicle having a diameter between 20-300nm (e.g., between 40-200 nm). Exosomes comprise membranes that encapsulate an internal space (i.e., a cavity), and in some aspects, can be produced from cells (e.g., production cells) by direct plasma membrane budding or by late endosomal fusion to the plasma membrane. In certain aspects, the exosomes comprise a scaffold moiety. As described below, exosomes may be derived from a producer cell and isolated from the producer cell based on their size, density, biochemical parameters, or a combination thereof. In some aspects, the exosomes of the present disclosure are produced by a cell expressing one or more transgene products. In some aspects, the term exosome refers to a population of exosomes.

In some aspects, an EV (e.g., exosome, e.g., nanovesicle) of the present disclosure is engineered by chemically attaching at least one biologically active molecule (e.g., a protein, such as an antibody or ADC, RNA or DNA (such as an antisense oligonucleotide), a small molecule drug, a toxin, PROTAC, AAV or morpholine) to the EV (e.g., exosome, e.g., nanovesicle) via a maleimide moiety. In some aspects, the maleimide moiety is part of a bifunctional reagent.

In some aspects, EVs (e.g., exosomes) or nanovesicles of the present disclosure may comprise various macromolecular payloads within an interior space (i.e., cavity), displayed on the exterior (exterior) surface or interior (cavity) surface of the EV, and/or across the membrane. In some aspects, the payload can include, for example, a nucleic acid, a protein, a carbohydrate, a lipid, a small molecule, and/or combinations thereof. In certain aspects, the EV (e.g., exosomes) comprise a scaffold moiety, such as a scaffold X protein or fragment thereof. EVs (e.g., exosomes) may be derived from living or dead organisms, explanted tissues or organs, prokaryotic or eukaryotic cells, and/or cultured cells. In some aspects, the EV (e.g., exosomes) are produced by cells expressing one or more transgene products. In other aspects, EVs of the present disclosure are, but are not limited to, nanovesicles, microsomes, microvesicles, exosomes, or apoptotic bodies.

As used herein, the term "fragment" of a protein (e.g., a biologically active molecule such as a therapeutic protein or a scaffold protein (such as a scaffold X protein or fragment thereof or a scaffold Y protein or fragment thereof)) refers to a protein having an amino acid sequence that is shorter than the naturally occurring sequence, N-terminal and/or C-terminal deletions, or any partial deletion of the protein, as compared to the naturally occurring protein.

As used herein, the term "functional fragment" refers to a protein fragment that retains the function of the protein. Thus, in some aspects, a functional fragment of a scaffold protein (e.g., a fragment of a scaffold X protein) retains the ability to attach or attach (e.g., via a maleimide moiety) a moiety (e.g., a biologically active molecule) on the luminal or outer surface of an EV (e.g., an exosome). Similarly, in certain aspects, a functional fragment of the scaffold Y protein retains the ability to attach (e.g., via a maleimide moiety) a moiety (e.g., a bioactive molecule) on the luminal surface of an EV (e.g., an exosome).

Whether a fragment is a functional fragment can be assessed by any art-known method of determining the protein content of an EV (e.g., exosome), including western blotting, FACS analysis, and fusion of the fragment with an autofluorescent protein (such as, for example, GFP). In certain aspects, a functional fragment of a scaffold X protein retains, for example, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the ability of a naturally occurring scaffold X protein to attach (e.g., via a maleimide moiety) a biologically active molecule on the luminal or outer surface of an EV (e.g., an exosome).

As used herein, the term "linking" or "attaching" a bioactive molecule to a cavity or outer surface of an EV (e.g., exosome) of the present disclosure includes (i) chemically linking "or" conjugating "the bioactive molecule, e.g., via a chemical linker such as a maleimide moiety, and (ii) non-chemically linking the bioactive molecule, also referred to as" fusing or fusion "(e.g., via a peptide bond, an amino acid linker, and/or a scaffold protein) to the EV (e.g., exosome) or a portion of the scaffold protein localized on the cavity or outer surface of the EV (e.g., exosome).

As used herein, the term "fusing, fused, fusion" or "non-chemically linking" a bioactive molecule on the cavity or outer surface of an EV (e.g., exosome) of the present disclosure via, for example, a scaffold protein refers to linking the bioactive molecule to a portion of the scaffold molecule (e.g., protein) that is positioned on the cavity or outer surface of the EV (e.g., exosome), respectively. In some aspects, fusion between biologically active molecules can be via genetic fusion (i.e., chimeric expression).

As used herein, the terms "chemical linkage" and "conjugation" are used interchangeably, each referring to the covalent attachment of two or more moieties using chemical moieties (e.g., maleimide moieties), each moiety comprising, for example, an EV, a scaffold moiety, a biologically active moiety, one or more linkers, a targeting moiety, and/or a tropism moiety, or any combination thereof. Thus, a first moiety (e.g., a scaffold (such as a scaffold X protein) or a lipid (such as cholesterol)) will be "chemically linked" to a second moiety (e.g., a biologically active moiety) via a thioether bond formed by a reaction between a maleimide group present in one moiety and a thiol group present in the other moiety.

As used herein, the term "extracellular" may be used interchangeably with the terms "outer", "exterior", and "extracapsular", where each term refers to an extramembranous element that encapsulates an EV (e.g., exosomes). As used herein, the term "intracellular" may be used interchangeably with the terms "inner", "interior", and "intravesicular", where each term refers to an intramembranous element that encapsulates an EV (e.g., exosomes). The term "cavity" refers to the membrane interior space that encloses an EV (e.g., exosome). Thus, an intracavitary element of an EV (e.g., an exosome) may be referred to herein as "positioned in" or "in" a cavity.

As used herein, the term "homology" refers to the overall relatedness between polymer molecules, for example between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In general, the term "homology" means an evolutionary relationship between two molecules. Thus, two molecules that are homologous will have a common evolutionary ancestor. In the context of the present disclosure, the term homology encompasses identity and similarity.

In some aspects, polymer molecules are considered "homologous" to each other if at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the monomers in the polymer molecule are identical (identical monomers) or similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

In the context of the present disclosure, substitutions (even if they are referred to as amino acid substitutions) are made at the nucleic acid level, i.e. the substitution of an amino acid residue by an alternative amino acid residue is made by replacing the codon encoding the first amino acid with the codon encoding the second amino acid.

As used herein, the term "identity" refers to the overall monomer conservation between polymeric molecules, for example, between polypeptide molecules or polynucleotide molecules (e.g., DNA molecules and/or RNA molecules). The term "identical" without any additional qualifiers, e.g., protein a is identical to protein B, means that the sequences are 100% identical (100% sequence identity). Describing two sequences as, for example, "70% identical" is equivalent to describing them as having, for example, "70% sequence identity".

For example, calculation of percent identity of two polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second polypeptide sequences to achieve optimal alignment, and non-identical sequences can be disregarded for comparison purposes). In certain aspects, the length of sequences aligned for comparison purposes is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the length of a reference sequence. The amino acids at the corresponding amino acid positions are then compared.

When a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, then the molecules at that position are identical. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, and gaps need to be introduced to achieve optimal alignment of the two sequences, taking into account the number of gaps and the length of each gap. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

Suitable software programs are available from a variety of sources and can be used for alignment of both protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST program suite (suite of programs) available from the National Center for Biotechnology Information BLAST website (blast.ncbi.n.lm.nih.gov.) of the united states government. Bl2seq uses the BLASTN or BLASTP algorithm between two sequences for comparison. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or mather, part of the EMBOSS suite of bioinformatics programs, and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk/Tools/psa.

Sequence alignments can be performed using methods known in the art, such as MAFFT, Clustal (Clustal W, X or Omega), MUSCLE, and the like.

Different regions within a single polynucleotide or polypeptide target sequence aligned with a polynucleotide or polypeptide reference sequence may each have their own percentage of sequence identity. Note that the percentage sequence identity values are rounded to the tenth position. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It should also be noted that the length value will always be an integer.

In certain aspects, the calculated formula for percent identity (% ID) of a first amino acid sequence (or nucleic acid sequence) to a second amino acid sequence (or nucleic acid sequence) is% ID ═ 100 × (Y/Z), where Y is the number of amino acid residues (or nucleobases) scored as identical matches in the first and second sequence alignments (e.g., by visual inspection or special sequence alignment programs) and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

One skilled in the art will appreciate that the generation of sequence alignments for calculating percent sequence identity is not limited to binary sequence-to-sequence comparisons driven by only primary sequence data. It is also understood that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystallographic protein structure), functional data (e.g., location of mutations), or phylogenetic data. Suitable programs for integrating the isomeric data to generate multiple sequence alignments are available at www.tcoffee.org, and alternatively can be, for example, T-Coffee available from EBI. It will also be appreciated that the final alignment used to calculate percent sequence identity may be automatically or manually assisted (cured).

As used herein, the term "immunomodulator" refers to an agent that acts on a target (e.g., a target cell) in contact with an EV (e.g., an exosome) and modulates the immune system. Non-limiting examples of immune modulators that can incorporate EVs (e.g., exosomes) and/or producer cells include agents such as modulators of checkpoint inhibitors, ligands of checkpoint inhibitors, cytokines, derivatives thereof, or any combination thereof. Immunomodulatory agents can also include agonists, antagonists, antibodies, antigen binding fragments, polynucleotides (such as siRNA, miRNA, incrna, mRNA, or DNA), or small molecules. In some aspects of the disclosure, the bioactive molecule is an immunomodulator.

As used herein, "immune response" refers to a biological response in a vertebrate against foreign factors or abnormal cells (e.g., cancer cells) that protects an organism from these factors and the diseases they cause. The immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, Natural Killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules produced by any of these cells or the liver, including antibodies, cytokines, and complements, which results in the selective targeting, binding, destruction, and/or elimination from the vertebrate body of cells or tissues infected with invading pathogens, cancer cells, or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues. The immune response includes, for example, activation or suppression of T cells (e.g., effector T cells, Th cells, CD4+ cells, CD8+ T cells, or Treg cells), or activation or suppression of any other cell of the immune system (e.g., NK cells). Thus, an immune response may include a humoral immune response (e.g., mediated by B cells), a cellular immune response (e.g., mediated by T cells), or both a humoral and a cellular immune response. In some aspects of the disclosure, a bioactive molecule is a molecule capable of eliciting an immune response.

In some aspects, the immune response is an "inhibitory" immune response. An inhibitory immune response is an immune response that blocks or attenuates the action of a stimulus (e.g., an antigen). In certain aspects, the inhibitory immune response comprises the production of inhibitory antibodies against the stimulus. In some aspects, the immune response is a "stimulatory" immune response. A stimulatory immune response is an immune response that results in the production of effector cells (e.g., cytotoxic T lymphocytes) that can destroy and clear a target antigen (e.g., a tumor antigen or virus).

As used herein, the term "immunoconjugate" refers to a compound comprising a binding molecule (e.g., an antibody) and one or more moieties (e.g., therapeutic or diagnostic moieties) chemically conjugated to the binding molecule. Typically, immunoconjugates are defined by the general formula: a- (LM) n, wherein a is a binding molecule (e.g., an antibody), L is an optional linker, M is a heterologous moiety that can be, for example, a therapeutic agent, a detectable label, etc., and n is an integer. In some aspects, multiple heterologous moieties can be chemically conjugated to different points of attachment in the same binding molecule (e.g., antibody). In other aspects, multiple heterologous moieties can be linked in series and to a point of attachment in a binding molecule (e.g., an antibody). In some aspects, multiple heterologous moieties (the same or different) can be conjugated to a binding molecule (e.g., an antibody).

Immunoconjugates can also be defined by the general formula in reverse order. In some aspects, the immunoconjugate is an "antibody-drug conjugate" ("ADC"). In the context of the present disclosure, the term "immunoconjugate" is not limited to chemically or enzymatically conjugated molecules. The term "immunoconjugate" as used in the present disclosure also includes genetic fusions. In some aspects of the disclosure, the biologically active molecule is an immunoconjugate.

As used herein, the terms "isolated," "purified," "extracted," and grammatical variations thereof, are used interchangeably to refer to the state of a preparation of a desired EV (e.g., a plurality of EVs of known or unknown quantity and/or concentration) that has been subjected to one or more purification processes (e.g., selection or enrichment of the desired EV (e.g., exosomes)). In some aspects, isolation or purification as used herein is a process of removing, partially removing (e.g., a portion of) EV (e.g., exosomes) from a sample containing producer cells. In some aspects, the isolated EV (e.g., exosome) composition has no detectable undesirable activity, or alternatively, the level or amount of undesirable activity is equal to or lower than an acceptable level or amount. In other aspects, an isolated EV (e.g., exosome) composition has an amount and/or concentration of a desired EV (e.g., exosome) equal to or greater than an acceptable amount and/or concentration. In other aspects, the isolated EV (e.g., exosome) composition is enriched compared to the starting material (e.g., producer cell preparation) from which the composition was obtained. Such enrichment may be enriched by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, at least about 99.99%, at least about 99.999%, at least about 99.9999%, or greater than 99.9999% as compared to the starting material. In some aspects, the isolated EV (e.g., exosome) preparation is substantially free of residual biological products. In some aspects, an isolated EV (e.g., exosome) preparation is 100% free, at least about 99% free, at least about 98% free, at least about 97% free, at least about 96% free, at least about 95% free, at least about 94% free, at least about 93% free, at least about 92% free, at least about 91% free, or at least about 90% free of any contaminating biological substances. The residual biological products may include non-biological materials (including chemicals) or undesirable nucleic acids, proteins, lipids, or metabolites. Substantially free of residual biological product may also mean that the EV (e.g., exosome) composition contains no detectable producer cells, and only the EV (e.g., exosomes) is detectable.

As used herein, the term "cavity-engineered EV" refers to an EV (e.g., exosome) whose membrane cavity surface or composition of the cavity is modified such that the cavity surface or cavity of the engineered EV (e.g., exosome) is different from the EV (e.g., exosome) or naturally-occurring EV (e.g., exosome) prior to modification.

The engineering may be directly within the cavity (i.e., void within the EV) or in the membrane of the EV (e.g., exosome), particularly at the cavity surface of the EV, such that the cavity and/or cavity surface of the EV (e.g., exosome) is altered. For example, the membrane is modified in its composition of proteins, lipids, small molecules, carbohydrates, etc., such that the cavity surface of the EV (e.g., exosomes) is modified. Similarly, the contents of the cavity may be modified. The composition may be altered by chemical, physical or biological means, or by production from cells previously modified by chemical, physical or biological means. In particular, the composition may be altered by genetic engineering, or by production from a cell previously modified by genetic engineering. In some aspects, a cavity-engineered EV (e.g., a cavity-engineered exosome) comprises an exogenous protein (i.e., a protein not naturally expressed by the EV (e.g., exosome)) or fragment or variant thereof, which may be exposed on the cavity surface or cavity of the EV (e.g., exosome), or a moiety may be attached to the cavity surface of the EV (e.g., exosome), attached to the EV. In other aspects, a lumen-engineered EV (e.g., a lumen-engineered exosome) comprises a higher-expressing native EV (e.g., exosome), a protein (e.g., scaffold X or scaffold Y), or a fragment or variant thereof, which may be a lumen exposed to the EV (e.g., exosome), or a moiety may be attached to a lumen surface of the EV (e.g., exosome).

As used herein, the term "macromolecule" refers to a nucleic acid, protein, lipid, carbohydrate, metabolite, or combination thereof.

As used herein, the term "maleimide moiety" or "MM" refers to a chemical moiety that links an EV (e.g., exosome) to a linker or biologically active molecule, and comprises a maleimide group:

where denotes the point of attachment to any thiol group on the EV (e.g. exosome) (e.g. free thiol present in the scaffold X protein) and the wavy line denotes the attachment site to the rest of the maleimide moiety.

In some aspects, indicates the point of attachment to any thiol group on an antibody, PROTAC, or any other biologically active molecule, and the wavy line indicates the attachment site (e.g., scaffold X protein) of the remainder of the maleimide moiety to an EV (e.g., exosome).

As used herein, the term "macromolecule" refers to a nucleic acid, protein, lipid, carbohydrate, metabolite, or combination thereof.

The term "modified," when used in the context of an EV (e.g., exosome) described herein, refers to an alteration or engineering of the EV (e.g., exosome) and/or its producer cell such that the modified EV (e.g., exosome) is different from the naturally-occurring EV (e.g., exosome). In some aspects, a modified EV (e.g., exosome) described herein comprises a membrane that differs in the composition of proteins, lipids, small molecules, carbohydrates, etc., as compared to the membrane of a naturally-occurring EV (e.g., exosome). For example, the membrane comprises a higher density or number of native EVs (e.g., exosomes), proteins, and/or the membrane comprises proteins that are not naturally present in EVs (e.g., exosomes). In certain aspects, such modifications to the membrane alter the outer surface of the EV (e.g., exosomes) (e.g., surface engineered EVs and exosomes described herein). In certain aspects, such modifications to the membrane alter the cavity surface of the EV (e.g., exosomes) (e.g., cavity engineered EVs and exosomes described herein).

As used herein, the term "modified protein" or "protein modification" refers to a protein having at least about 15% identity to the non-mutated amino acid sequence of the protein. Modifications of the protein include fragments or variants of the protein. Modifications of a protein may also include chemical or physical modifications to fragments or variants of the protein.

As used herein, the terms "modulate," "modify," and grammatical variations thereof, when applied to a particular concentration, level, expression, function, or behavior, generally refer to the ability to change by increasing or decreasing, e.g., directly or indirectly promoting/stimulating/upregulating or interfering/inhibiting/downregulating, e.g., as an antagonist or agonist, the particular concentration, level, expression, function, or behavior. In some cases, a modulator may increase and/or decrease a certain concentration, level, activity, or function relative to a control, or relative to a generally expected average level of activity, or relative to a control activity level.

As used herein, the term "nanovesicle" refers to an extracellular vesicle having a diameter between about 20nm and about 250nm (e.g., between about 30nm and about 150 nm) and is produced from a cell (e.g., a production cell) by direct or indirect manipulation such that the unmanipulated cell does not produce nanovesicles. Suitable manipulations of the cells to produce nanovesicles include, but are not limited to, continuous extrusion, treatment with an alkaline solution, sonication, or combinations thereof. In some aspects, the production of nanovesicles can result in the destruction of the producer cell. In some aspects, the population of nanovesicles described herein is substantially free of vesicles derived from cells by direct budding from the plasma membrane or late endosome fusion to the plasma membrane. In certain aspects, the nanovesicles comprise a scaffold moiety, such as a scaffold X protein or fragment thereof and/or a scaffold Y protein or fragment thereof. Once the nanovesicles are derived from the producer cell, they can be isolated from the producer cell based on their size, density, biochemical parameters, or a combination thereof.

As used herein, the term "payload" refers to a bioactive molecule (e.g., a therapeutic agent) that acts on a target (e.g., a target cell) contacted with an EV (e.g., an exosome) of the present disclosure. Non-limiting examples of payloads that can be introduced into an EV (e.g., exosomes) include therapeutic agents such as nucleotides (e.g., nucleotides comprising a detectable moiety or toxin or that disrupt transcription), nucleic acids (e.g., DNA or mRNA molecules encoding polypeptides such as enzymes, or RNA molecules with regulatory functions such as miRNA, dsDNA, incrna, and siRNA), amino acids (e.g., comprising a detectable moiety or toxin or that disrupt translation), polypeptides (e.g., enzymes), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins). In certain aspects, the payload comprises an antigen. As used herein, the term "antigen" refers to any agent that elicits an immune response (cellular or humoral) against itself when introduced into a subject. In some aspects, the antigen is used to elicit an immune response, i.e., as a vaccine. In other aspects, the payload includes an adjuvant. In some aspects, the payload molecule is covalently attached to the EV (e.g., exosome) via a maleimide moiety.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" and grammatical variations thereof encompasses any agent approved by the U.S. federal regulatory agency or listed in the U.S. pharmacopeia for use in animals, including humans, and any carrier or diluent that does not cause undesirable physiological effects to the extent that administration of the composition to a subject is prohibited and does not abrogate the biological activity and properties of the administered compound. Including excipients and carriers that may be used in the preparation of pharmaceutical compositions, which are generally safe, non-toxic and desirable.

As used herein, the term "pharmaceutical composition" refers to one or more of the compounds described herein, e.g., EVs, such as exosomes of the present disclosure, mixed or blended with or suspended in one or more other chemical components (such as pharmaceutically acceptable carriers and excipients). One purpose of the pharmaceutical composition is to facilitate administration of an EV preparation (e.g., exosomes) to a subject in need thereof.

As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. The term refers to the primary structure of the molecule. Thus, the term includes triple-, double-and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double-and single-stranded ribonucleic acid ("RNA"). It also includes modified, e.g., by alkylation and/or by capping, as well as unmodified polynucleotide forms. In particular, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including trnas, rrnas, hrnas, sirnas and mrnas, whether spliced or not, any other type of polynucleotide (which is an N-or C-glycoside of a purine or pyrimidine base), as well as other polymers containing a positive nucleotide backbone, such as polyamides (e.g., peptide nucleic acids "PNAs") and poly-morpholine polymers, as well as other synthetic sequence-specific nucleic acid polymers (provided that the polymer contains nucleobases with a configuration that allows base pairing and base stacking, such as found in DNA and RNA). In some aspects of the disclosure, the biologically active molecule attached to the EV (e.g., exosome) via a maleimide moiety is a polynucleotide, such as an antisense oligonucleotide. In a particular aspect, the polynucleotide comprises mRNA. In other aspects, the mRNA is a synthetic mRNA. In some aspects, the synthetic mRNA comprises at least one non-natural nucleobase. In some aspects, all of a class of nucleobases have been replaced with a non-natural nucleobase (e.g., all of the uridines in a polynucleotide disclosed herein can be replaced with a non-natural nucleobase, such as a 5-methoxyuridine). In some aspects of the disclosure, the biologically active molecule is a polynucleotide.

In some aspects, the polynucleotides disclosed herein can be modified to introduce a thiol group that can be used to react with a maleimide moiety. In some aspects, the polynucleotides disclosed herein can be modified to introduce a maleimide moiety group that is available for reaction with a thiol group.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may comprise modified amino acids. These terms also encompass amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (such as conjugation to a labeling element). The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. In some aspects of the disclosure, a biologically active molecule attached to an EV (e.g., exosome) via a maleimide moiety is a polypeptide, e.g., an antibody or derivative thereof, such as an ADC, PROTAC, toxin, fusion protein, or enzyme.

As used herein, the term "polypeptide" refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. The polypeptide may be a single polypeptide or may be a multi-molecular complex, such as a dimer, trimer or tetramer. They may also include single-or multi-chain polypeptides. The most common disulfide bonds are present in multi-chain polypeptides. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding naturally occurring amino acid. In some aspects, a "peptide" may be less than or equal to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

In some aspects, the polypeptides disclosed herein can be modified to introduce a thiol group that can be used to react with a maleimide moiety. In some aspects, the polypeptides disclosed herein can be modified to introduce a maleimide moiety that is available for reaction with a thiol group.

As used herein, the term "preventing" and variations thereof refers to delaying the onset of a disease, disorder, and/or condition, either partially or completely; partially or completely delaying the onset of one or more symptoms, features, or clinical manifestations of a particular disease, disorder, and/or condition; delay, partially or completely, the onset of one or more symptoms, features, or manifestations of a particular disease, disorder, and/or condition; delay in progression of a particular disease, disorder, and/or condition, either partially or completely; and/or reducing the risk of developing a pathology associated with the disease, disorder, and/or condition. In some aspects, prevention of outcome is achieved by prophylactic treatment.

As used herein, the term "producer cell" refers to a cell used to produce an EV (e.g., exosome). The producer cells may be cells cultured in vitro or in vivo. Production cells include, but are not limited to, cells known to be capable of efficiently producing EV (e.g., exosomes), e.g., HEK293 cells, Chinese Hamster Ovary (CHO) cells, Mesenchymal Stem Cells (MSC), BJ human foreskin fibroblasts, fHDF fibroblasts, recombinant human embryonic stem cells (hEV), recombinant human embryonic stem cells (hMC), recombinant human embryonic stem cells (hGH), recombinant human embryonic stem cells (hD), recombinant human embryonic stem cells (hGH (hD), recombinant human embryonic stem cells (hD), and recombinant human embryonic stem cells (hD) cells (hD) and recombinant human embryonic stem cells (hD) comprising such as recombinant human embryonic stem cells,Neuronal precursor cells,Amniotic fluid cells, adipose-derived mesenchymal stem cells, and RPTEC/TERT1 cells. In certain aspects, the producer cell is not an antigen presenting cell. In some aspects, the producer cell is not a dendritic cell, a B cell, a mast cell, a macrophage, a neutrophil, a Kupffer-Browicz cell, a cell derived from any of these cells, or any combination thereof.

As used herein, "prophylactic" refers to a treatment or course of action for preventing the onset of a disease or condition, or preventing or delaying the symptoms associated with a disease or condition.

As used herein, "prevention" refers to measures taken to maintain health and prevent or delay the onset of bleeding episodes, or to prevent or delay symptoms associated with a disease or condition.

A "recombinant" polypeptide or protein refers to a polypeptide or protein produced by recombinant DNA techniques. Recombinantly produced polypeptides and proteins expressed in engineered host cells isolated for the purposes of this disclosure are considered natural or recombinant polypeptides that have been isolated, fractionated or partially or substantially purified by any suitable technique. The polypeptides disclosed herein can be recombinantly produced using methods known in the art. Alternatively, the proteins and peptides disclosed herein can be chemically synthesized. In some aspects of the disclosure, a scaffold X protein and/or a scaffold Y protein present in an EV (e.g., exosome) may be recombinantly produced by overexpressing the scaffold protein in a producer cell, such that the level of scaffold protein in the resulting EV (e.g., exosome) is significantly increased relative to the level of scaffold protein present in an EV (e.g., exosome) of a producer cell that does not overexpress such scaffold protein.

As used herein, the term "scaffold moiety" refers to a molecule, e.g., a protein or fragment thereof (e.g., a functional fragment thereof), on a luminal surface (such as a scaffold Y protein) or an outer surface (such as a scaffold X protein) of an EV (e.g., exosomes) that can be used to attach a payload (e.g., a biologically active molecule) or any other compound of interest (e.g., AAV) to the EV (e.g., exosomes). In some aspects, a scaffold protein is a polypeptide that does not naturally occur in an EV (e.g., an exosome). In certain aspects, the scaffold moiety comprises a synthetic molecule. In some aspects, the scaffold moiety comprises a non-polypeptide moiety. In other aspects, the scaffold moiety comprises, for example, a lipid, carbohydrate, protein, or combination thereof (e.g., a glycoprotein or proteolipid) naturally occurring in an EV (e.g., exosome). In some aspects, the scaffold moiety comprises a lipid, carbohydrate, or protein that is not naturally present in an EV (e.g., exosome). In some aspects, the scaffold moiety comprises a lipid or carbohydrate that is naturally present in the EV (e.g., exosome), but has been enriched in the EV (e.g., exosome) relative to the basal/native/wild-type levels. In some aspects, the scaffold moiety comprises a protein that is naturally present in the EV (e.g., exosome), but has been enriched in the EV (e.g., exosome) relative to basal/native/wild-type levels, e.g., by recombinant overexpression in a producer cell. In certain aspects, the scaffold moiety is a scaffold X protein or a fragment thereof. In some aspects, the scaffold moiety is a scaffold Y protein or a fragment thereof. In other aspects, the EV comprises a scaffold X protein or fragment thereof and a scaffold Y protein or fragment thereof.

As used herein, the term "scaffold X" refers to an EV (e.g., exosome) protein that has been identified on the surface of an EV (e.g., exosome), and may be engineered to be overexpressed in the EV. See, for example, U.S. patent No. 10,195,290, which is incorporated by reference herein in its entirety. Non-limiting examples of scaffold X proteins include: prostaglandin F2 receptor negative regulator ("PTGFRN"); basic immunoglobulin ("BSG"); immunoglobulin superfamily member 2 ("IGSF 2"); immunoglobulin superfamily member 3 ("IGSF 3"); immunoglobulin superfamily member 8 ("IGSF 8"); integrin beta-1 ("ITGB 1"); integrin α -4 ("ITGA 4"); 4F2 cell surface antigen heavy chain ("SLC 3a 2"); and a class of ATP transporters ("ATP 1a 1", "ATP 1a 2", "ATP 1 A3", "ATP 1a 4", "ATP 1B 3", "ATP 2B 1", "ATP 2B 2", "ATP 2B 3", "ATP 2B"), fragments thereof, and any combination thereof. In some embodiments, the scaffold X protein may be an intact protein or a fragment thereof (e.g., a functional fragment, e.g., the smallest fragment that is capable of linking another moiety to the outer surface of an EV (e.g., exosome) or to the surface of a lumen). In some aspects, the scaffold X may attach a bioactive molecule to the outer surface or cavity of an EV (e.g., exosome). In some aspects of the disclosure, the bioactive molecule may be chemically linked to the scaffold X protein or fragment thereof via a maleimide moiety. In some aspects, a bioactive molecule can be chemically attached to a scaffold X protein or fragment thereof via a maleimide moiety on the luminal surface of an EV (e.g., exosome). Non-limiting examples of other stent portions that may be used in the present disclosure include: aminopeptidase N (CD 13); enkephalinase (membrane metalloendopeptidase; MME); ectonucleotide pyrophosphatase/phosphodiesterase family member 1(ENPP 1); neuropilin-1 (NRP 1); CD9, CD63, CD81, PDGFR, GPI protein, lactadherin, LAMP2 and LAMP2B, fragments thereof, and any combination thereof.

In some aspects, a scaffold moiety (e.g., an EV protein described in U.S. patent No. 10,195,290, which is incorporated herein by reference in its entirety) forms a fusion with a binding partner (e.g., an antigen binding domain, a capsid protein, an Fc receptor, a binding partner of a chemically-induced dimer, or any combination thereof) that can be used to bind another molecule (i.e., a second binding partner).

As used herein, the term "binding partner" refers to one member of at least two elements that interact to form a multimer (e.g., a dimer). In some aspects, the binding partner is a first binding partner that interacts with a second binding partner. In some aspects, the binding partner is a first binding partner that interacts with the second binding partner and/or the third binding partner. Any binding partner can be used in the compositions and methods disclosed herein. In some aspects, the binding partner can be a polypeptide, a polynucleotide, a fatty acid, a small molecule, or any combination thereof. In certain aspects, the binding partners (e.g., the first binding partner and/or the second binding partner) are selected from chemically-induced dimeric first and second binding partners.

As used herein, the term "scaffold Y" refers to an EV (e.g., exosome) protein that has been identified within the lumen of an EV (e.g., exosome), and may be engineered to be overexpressed in the EV. See, for example, international application No. PCT/US2018/061679, which is incorporated herein by reference in its entirety. Non-limiting examples of scaffold Y proteins include: myristoylated alanine-rich protein kinase C substrate ("MARCKS"); myristoylated alanine-rich protein kinase C substrate like 1 ("MARCKSL 1"); and brain acid soluble protein 1 ("BASP 1"), fragments thereof, and any combination thereof. In some aspects, a scaffold Y protein may be an intact protein or a fragment thereof (e.g., a functional fragment, e.g., a minimal fragment capable of attaching a moiety to the luminal surface of an EV (e.g., an exosome)). In some aspects, a scaffold Y protein or fragment thereof may attach a moiety to the luminal surface of an EV (e.g., exosome). In some aspects of the disclosure, a moiety (e.g., a biologically active molecule) may be linked (e.g., chemically linked) to a scaffold Y protein or fragment thereof. In some aspects, a moiety (e.g., a bioactive molecule) may be attached (e.g., chemically attached) to a scaffold Y protein or fragment thereof on the luminal surface of an EV (e.g., an exosome).

In certain aspects, the scaffold protein comprises a fragment of an EV protein. In some aspects, the scaffold protein comprises a fragment of MARCKS, MARCKSL1, or BASP 1. In some aspects, the scaffold protein comprises the amino acid sequence GGKLSKK (SEQ ID NO: 17). In some aspects, the scaffold protein comprises the amino acid sequence GGKLSKK (SEQ ID NO:17) wherein the C-terminal glycine residue is myristoylated.

In some aspects, the scaffold protein is a transmembrane protein. As used herein, a "transmembrane protein" refers to any protein comprising an extracellular domain (e.g., at least one amino acid located outside, e.g., extracapsular, EV (e.g., exosome) membrane), a transmembrane domain (e.g., at least one amino acid located inside, e.g., exosome membrane) and an intracellular domain (e.g., at least one amino acid located inside, e.g., exosome membrane). In some aspects, a scaffold protein described herein is a type I transmembrane protein, wherein the N-terminus of the transmembrane protein is positioned in an extracellular space, e.g., outside of the membrane, e.g., extracapsular, that encapsulates an EV (e.g., exosome). In some aspects, a scaffold protein described herein is a type II transmembrane protein, wherein the N-terminus of the transmembrane protein is located in an intracellular space, e.g., within a membrane that encapsulates an EV (e.g., an exosome), e.g., on the luminal side of the membrane, e.g., within the vesicle.

As used herein, the term "self-immolative spacer" refers to a spacer as defined below: i.e., if the bond of the spacer to the first moiety (e.g., cleavable linker) is cleaved, it will spontaneously separate from the second moiety (e.g., biologically active molecule).

As used herein, the term "similarity" refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. The calculation of percent similarity of polymer molecules to each other can be performed in the same manner as the calculation of percent identity, except that the calculation of percent similarity takes into account conservative substitutions as understood in the art. It is understood that the percent similarity depends on the comparison scale used, i.e., whether amino acids are compared, e.g., according to their evolutionary proximity, charge, volume, flexibility, polarity, hydrophobicity, aromaticity, isoelectric point, antigenicity, or a combination thereof.

As used herein, the term "spacer" refers to a bifunctional chemical moiety capable of covalently linking two spaced apart moieties (e.g., a cleavable linker and a bioactive molecule) together to form a generally stable binary molecule.

Unless otherwise indicated, reference to a compound having one or more stereocenters is intended to refer to each stereoisomer and all combinations of stereoisomers thereof.

The terms "subject," "patient," "individual," and "host" and variations thereof are used interchangeably herein to refer to any mammalian subject in need of diagnosis, treatment, or therapy, including, but not limited to, humans, livestock (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, and the like), particularly humans. The methods described herein are suitable for human therapy and veterinary applications.

As used herein, the term "substantially free" means that the sample comprising the EV (e.g., exosomes) comprises macromolecules (e.g., contaminants) at a concentration of less than 10% as a percent mass/volume (m/v). Some fractions may contain less than about 0.001%, less than about 0.01%, less than about 0.05%, less than about 0.1%, less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, less than about 0.6%, less than about 0.7%, less than about 0.8%, less than about 0.9%, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, or less than about 10% (m/v) of macromolecules.

As used herein, the term "surface engineered EV" (e.g., scaffold X engineered exosomes) refers to EVs whose membranes or surface is compositionally modified such that the surface of the engineered EV is different from the EV or naturally occurring EV prior to modification.

As used herein, the term "surface engineered exosomes" (e.g., scaffold X engineered exosomes) refers to exosomes whose membrane or surface (outer surface or lumen surface) is compositionally modified such that the surface of the engineered exosomes is different from the exosomes prior to modification or naturally occurring exosomes.

The engineering may be on the surface of the EV (e.g., exosome), or in the membrane of the EV (e.g., exosome), such that the surface of the EV (e.g., exosome) is altered. For example, the composition of the membrane, such as proteins, lipids, small molecules, carbohydrates, or combinations thereof, may be modified. The composition may be altered by chemical, physical or biological means, or by production from cells previously or simultaneously modified by chemical, physical or biological means. In particular, the composition may be altered by genetic engineering, or by production from a cell previously modified by genetic engineering. In some aspects, a surface engineered EV (e.g., exosome) comprises an exogenous protein (i.e., a protein not naturally expressed by the EV (e.g., exosome)) or fragment or variant thereof, which may be exposed to the surface of the EV (e.g., exosome), or a moiety may be attached to the surface of the EV (e.g., exosome). In other aspects, a surface engineered EV (e.g., exosome) comprises a higher expression (e.g., higher number) of a native EV (e.g., exosome) protein (e.g., scaffold X protein) or fragment or variant thereof, which may be exposed to the surface of the EV (e.g., exosome), or a moiety may be attached to the surface of the EV (e.g., exosome). In a particular aspect, a surface engineered EV (e.g., exosome) comprises a modification of one or more membrane components, e.g., a protein (such as a scaffold X protein or fragment thereof), a lipid, a small molecule, a carbohydrate, or any combination thereof, wherein at least one component is linked (e.g., chemically linked, e.g., via a maleimide moiety) to a bioactive molecule.

As used herein, the term "therapeutically effective amount" is an amount of an agent or pharmaceutical compound comprising an EV or exosome of the present disclosure sufficient to produce a desired therapeutic, pharmacological and/or physiological effect in a subject in need thereof. A therapeutically effective amount may be a "prophylactically effective amount" since prophylaxis may be considered treatment.

As used herein, the term "treatment" or "treating" refers to, for example, a reduction in the severity of a disease or condition; shortening the duration of the disease course; amelioration or elimination of one or more symptoms associated with a disease or condition; providing a beneficial effect to a subject having a disease or condition, but not necessarily curing the disease or condition; or any combination thereof. The term also includes the defense or prevention of the disease or condition or symptoms thereof. In one aspect, the term "treating" or "treatment" refers to inducing an immune response against an antigen in a subject in need thereof, e.g., by administering an EV (e.g., exosome) comprising an antigen (vaccine antigen) and optionally an adjuvant on the outer surface of the EV (e.g., exosome).

As used herein, the term "variant" of a molecule (e.g., a functional molecule, antigen, adjuvant, scaffold X protein or fragment thereof, and/or scaffold Y protein or fragment thereof) refers to a molecule that shares some structural and functional identity with another molecule, when compared by methods known in the art. For example, a variant of one protein may include a substitution, insertion, deletion, frameshift, or rearrangement in another protein.

In some aspects, the variant or derivative of scaffold X comprises a scaffold X variant having at least about 70% identity to a full-length, mature PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3a2, or ATP transporter, or a fragment (e.g., a functional fragment) of PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3a2, or ATP transporter.

In some aspects, a variant or fragment variant of a scaffold X protein disclosed herein or a derivative thereof retains the ability to specifically target an EV (e.g., an exosome). In some aspects, the scaffold X or scaffold X derivative comprises one or more mutations, such as conservative amino acid substitutions.

In some aspects, the variant of scaffold Y or derivative thereof comprises a variant having at least 70% identity to MARCKS, MARCKSL1, BASP1, or a fragment of MARCKS, MARCKSL1, or BASP 1.

In some aspects, a variant or fragment variant of the scaffold Y protein or derivative thereof retains the ability to specifically target the luminal surface of an EV (e.g., an exosome). In some aspects, the scaffold Y comprises one or more mutations, such as conservative amino acid substitutions.

Naturally occurring variants are referred to as "allelic variants" and refer to one of several alternative forms of a gene occupying a given locus on a chromosome of an organism (Genes II, Lewin, b., editors, John Wiley & Sons, New York (1985)). These allelic variants may vary at the polynucleotide and/or polypeptide level and are encompassed by the present disclosure. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.

Variants can be produced to improve or alter the characteristics of the polypeptide using known methods of protein engineering and recombinant DNA technology. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function. Ron et al, J.biol.chem.268:2984-2988(1993) (herein incorporated by reference in its entirety) report that variant KGF proteins have heparin binding activity even after deletion of 3, 8 or 27 amino-terminal amino acid residues. Similarly, interferon gamma shows up to ten-fold activity after deletion of 8-10 amino acid residues from the carboxy-terminus of the protein. (Dobeli et al, J.Biotechnology 7:199-216(1988), herein incorporated by reference in its entirety).

Furthermore, there is a large body of evidence that variants generally retain biological activity similar to naturally occurring proteins. For example, Gayle and colleagues (J.biol.chem 268:22105-22111(1993), incorporated herein by reference in its entirety) have conducted extensive mutation analysis on the human cytokine IL-1 a. They generated 3,500 individual IL-1a mutants using random mutagenesis, with an average of 2.5 amino acid changes for each variant over the entire length of the molecule. Multiple mutations were examined at each possible amino acid position. Researchers have found that "most molecules can be altered with little effect on [ binding or biological activity ]. "(see abstract). In fact, of the 3,500 nucleotide sequences examined, only 23 unique amino acid sequences produced proteins with significantly different activities from the wild type.

As described above, variants or derivatives include, for example, modified polypeptides. In some aspects, for example, variants or derivatives of polypeptides, polynucleotides, lipids, glycoproteins are the result of chemical and/or endogenous modifications. In some aspects, the variant or derivative is the result of an in vivo modification. In some aspects, the variant or derivative is the result of an in vitro modification. In still other aspects, the variant or derivative is the result of an intracellular modification in the producer cell.

Modifications present in variants and derivatives include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, covalent attachment of Glycosylphosphatidylinositol (GPI), hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Mei et al, Blood 116:270-79(2010), incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, prenylation, racemization, selenylation, sulfation, amidation, conjugation, etc, Transport RNA-mediated addition of amino acids to proteins, such as arginylation and ubiquitination.

In some aspects, the scaffold X protein and/or scaffold Y protein may be modified at any convenient location. In some aspects, the bioactive molecule can be modified at any convenient location. In particular aspects of the disclosure, EVs (e.g., exosomes), components (e.g., proteins such as scaffold X protein, scaffold Y protein, lipids, glycans, or combinations thereof) and/or biologically active molecules (e.g., antibodies or ADCs, PROTACs, small molecules such as cyclic dinucleotides, toxins such as MMAE, STING agonists, tolerizing agents, antisense oligonucleotides, antigens such as vaccine antigens, adjuvants, targeting moieties, tropism moieties, or any combination thereof) may be modified to produce derivatives comprising at least one maleimide moiety.

Conjugated EV (e.g., exosome)

Extracellular Vesicles (EV) typically have a diameter of 20nm to 1000 nm; for example, exosomes are small extracellular vesicles, typically 100 to 200nm in diameter. EV (e.g., exosomes) consists of a restricted lipid bilayer and a variety of proteins and nucleic acids (Maas, S.L.N. et al, trends.cell biol.27(3):172-188 (2017)). EVs (e.g., exosomes) exhibit preferential uptake in discrete cell types and tissues, and their tropism can be guided by the addition of proteins on their surface that interact with receptors on the surface of target cells (Alvarez-Erviti, l. et al, nat. biotechnol.29(4): 341-.

Unlike antibodies, EVs (e.g., exosomes) can accommodate a large number of molecules attached to their surface, with each EV (e.g., exosome) being on the order of thousands to tens of thousands of molecules. Thus, EV (e.g., exosome) -drug conjugates represent a platform that can deliver high concentrations of therapeutic compounds to discrete cell types while limiting overall systemic exposure to the compounds, thereby reducing off-target toxicity.

The present disclosure provides EVs (e.g., exosomes) designed by reacting a first molecular entity comprising a free thiol group with a second molecular entity comprising a maleimide group, wherein the maleimide moiety covalently links the first molecular entity (e.g., EV, e.g., exosome or component thereof (e.g., scaffold X protein or lipid)) to the second molecular entity (e.g., bioactive molecule) via the maleimide moiety, as shown in fig. 1A.

Non-limiting examples of biologically active molecules that can be linked to an EV (e.g., exosomes) via a maleimide moiety include agents such as nucleotides (e.g., nucleotides comprising a detectable moiety or toxin or disrupting transcription), nucleic acids (e.g., DNA or mRNA molecules encoding a polypeptide such as an enzyme, or RNA molecules with regulatory functions such as miRNA, dsDNA, lncRNA, or siRNA), morpholines, amino acids (e.g., amino acids comprising a detectable moiety or disrupting translation of a toxin), polypeptides (e.g., enzymes), lipids, carbohydrates, small molecules (e.g., small molecule drugs and toxins), antigens (e.g., vaccine antigens), adjuvants (e.g., vaccine adjuvants), and the like.

In some aspects, EVs (e.g., exosomes) of the present disclosure may comprise more than one type of bioactive molecule. In some aspects, the biologically active molecule can be, for example, a small molecule (such as a cyclic dinucleotide), a toxin (such as an auristatin (e.g., monoethyl auristatin E, MMAE)), an antibody (e.g., a naked antibody or antibody-drug conjugate), a STING agonist, a tolerant, an antisense oligonucleotide, PROTAC, morpholine, a lysophosphatidic acid receptor antagonist (e.g., LPA1 antagonist), or any combination thereof. In some aspects, an EV (e.g., exosome) of the present disclosure may comprise, for example, a vaccine antigen and optionally a vaccine adjuvant. In some aspects, an EV (e.g., exosome) of the present disclosure may comprise a therapeutic payload (e.g., STING or one of the payloads disclosed below) and a targeting moiety and/or a tropism moiety.

Thus, the methods disclosed herein can result in a molecular entity as shown in fig. 1A, wherein an EV (e.g., exosome) or any molecular component thereof, such as a polypeptide (e.g., scaffold X protein or fragment thereof), lipid, lipoprotein, glycoprotein, or any variant or derivative of a naturally-occurring or non-naturally-occurring protein, positioned on the EV (e.g., exosome), can be chemically linked to a biologically active molecule, e.g., a therapeutic payload, a targeting moiety, a tropism moiety, or any combination thereof, via a maleimide moiety. As shown in fig. 1A, in some aspects, an EV (e.g., exosome) comprising a thiol (thiol) group or molecular component thereof may be reacted with a maleimide group attached to a biologically active moiety. In other aspects, EVs (e.g., exosomes) comprising maleimide groups or molecular components thereof may react with thiol (thiol) groups present in the biologically active moiety. In both cases, the final product is a bioactive molecule chemically linked to the EV (e.g., exosome) via a thioether bond.

Ii.a. maleimide moiety

The maleimide moiety may be any chemical moiety comprising a maleimide group (e.g., a bifunctional chemical moiety that links an EV (e.g., exosome) to a linker, such as a peptide):

wherein

(i) Denotes the point of attachment to any available maleimide reactive group present on the EV (e.g. exosomes), e.g. a free thiol group of the scaffold X protein; and the number of the first and second electrodes,

(ii) the wavy line indicates the attachment site to the remainder of the maleimide moiety.

In some aspects, the maleimide moiety is attached to a sulfur atom attached to an EV (e.g., an exosome), such as a sulfur atom naturally occurring in a thiol group or a sulfur atom introduced via chemical modification or via mutation.

In some aspects, the maleimide moiety has formula (I):

wherein

(i)R¹Selected from the group consisting of: -C_1-10Alkylene-, -C_3-8Carbocycle-, -O- (C)_1-8Alkylene) -, -arylene-, -C_1-10Alkylene-arylene-, -arylene-C_1-10Alkylene-, -C_1-10Alkylene- (C)_3-8Carbocycle) -, - (C)_3-8Carbocyclic ring) -C_1-10Alkylene-, -C_3-8Heterocycle-, -C_1-10Alkylene- (C)_3-8Heterocycle) -, - (C)_3-8Heterocycle) -C_1-10Alkylene-, - (CH)₂CH₂O)_r-and- (CH)₂CH₂O)_r-CH₂-；

(ii) r is an integer, for example 1 to 10;

(iii) Denotes the point of attachment to any available reactive sulfur atom, e.g., the sulfur present in a thiol group on an EV (e.g., exosome); and the number of the first and second electrodes,

(iv) the wavy line represents the attachment site of the maleimide moiety to the biologically active molecule.

In some aspects, R¹is-C_1-8Alkylene-, -C_3-6Carbocycle-, -O- (C)_1-6Alkylene) -, -arylene-, -C_1-8Alkylene-arylene-, -arylene-C_1-8Alkylene-, -C_1-8Alkylene- (C)_3-6Carbocycle) -, - (C)_3-6Carbocyclic ring) -C_1-8Alkylene-, -C_3-6Heterocycle-, -C_1-8Alkylene- (C)_3-6Heterocycle) -, - (C)_3-6Heterocycle) -C_1-8Alkylene-, - (CH)₂CH₂O)_r-and- (CH)₂CH₂O)_r-CH₂-; wherein r is an integer, e.g., 1 to 10;

in some aspects, R¹Is- (CH)₂)_s-, cyclopentyl, cyclohexyl, -O- (CH)₂)_s-, phenyl, -CH₂-phenyl-, phenyl-CH₂-、-CH₂Cyclopentyl, cyclopentyl CH₂-、-CH₂-cyclohexyl, cyclohexyl-CH₂-、-(CH₂CH₂O)_r-, and- (CH)₂CH₂O)_r-CH₂-; where r is an integer, for example 1 to 6.

In some aspects, R¹Is- (CH)₂)_s-, where s is, for example, 4, 5 or 6.

In some aspects, the maleimide moiety has formula (II), wherein R¹Is- (CH)₂)₅-：

In some aspects, the maleimide moiety has formula (III), wherein R¹Is- (CH)₂CH₂O)_r-CH₂-, and wherein r is 2:

in some aspects, the maleimide moiety is covalently attached to a functional group present on the EV (e.g., exosome), wherein the functional group is a thiol (thiol) group. In one aspect, the sulfhydryl group is on a protein on the surface of an EV (e.g., exosome), such as a scaffold X protein or fragment or variant thereof. For example, in some aspects, a thiol group may be present on a thiol lipid, such as cholesterol-SH, DSPE-SH, or a derivative thereof, such as cholesterol-PEG-SH or DSPE-PEG-SH.

In other aspects, the maleimide moiety is covalently attached to a functional group present on the EV (e.g., exosome) that has been chemically derivatized to provide the maleimide moiety. For example, in one aspect, an amine functional group (e.g., lysine or arginine, or an amine on the side chain of the terminal amine group of a protein) present on an EV (e.g., exosome) may be derivatized with a bifunctional reagent comprising, for example, a succinimide moiety and a maleimide moiety.

In other aspects, a carboxyl functional group (e.g., glutamic acid or aspartic acid, or a carboxyl group on a side chain of a terminal carboxyl group of a protein) present on an EV (e.g., exosome) can be derivatized with a bifunctional reagent comprising, for example, an isocyanate moiety and a maleimide moiety. In still other aspects, a carbonyl group (oxidized carbohydrate) present on an EV (e.g., exosome) may be derivatized with a bifunctional reagent comprising, for example, a hydrazine moiety and a maleimide moiety.

In general, the methods disclosed herein can be performed using any reagent, such as a bifunctional or multifunctional reagent that, when reacted with a molecule (e.g., protein, lipid, sugar) present on the surface (outer surface or luminal surface) of an EV (e.g., exosome), will covalently or non-covalently modify the molecule to produce a modified molecule comprising at least one maleimide moiety. A molecule present on the surface (outer surface or luminal surface) of an EV (e.g., exosome) may be naturally occurring, or it may be non-naturally occurring, i.e., it has been modified, e.g., via chemical modification, incubated with a composition comprising a non-naturally occurring molecule, or via mutation (e.g., via mutation to introduce one or more cysteine amino acids into a protein).

Bifunctional reagents comprising a maleimide moiety, reagents in which a number of maleimide-containing units can be multimerized, or maleimide-containing reagents to which a functional moiety (e.g., PEG) can be added via a maleimide group include, for example, a bifunctional reagent comprising a hydrazine moiety and a maleimide moiety, a bifunctional reagent comprising an isocyanate moiety and a maleimide moiety, a bifunctional reagent comprising an N-hydroxysuccinimide ester moiety and a maleimide moiety, a bifunctional reagent comprising a succinimide moiety and a maleimide moiety, biotin-maleimide, streptavidin-maleimide, N-4-maleimidobutyric acid, N- (4-maleimidobutoxy) succinimide, N- [5- (3' -maleimidopropylamide) -1-carboxypentyl.]Iminodiacetic acid, maleimide-PEG-succinimidyl esters (e.g., maleimide-PEG₁₂-succinimidyl ester, maleimide-PEG₂-succinimidyl estermaleimide-PEG₂₀₀₀-succinimidyl ester, maleimide-PEG₅₀₀₀-succinimidyl ester, or maleimide-PEG_n-succinimidyl ester (wherein 1<n<5000) maleimide-PEG-maleimide (e.g., maleimide-PEG) ₁₂-maleimide, maleimide-PEG₂-maleimide, maleimide-PEG₂₀₀₀-maleimide, maleimide-PEG₅₀₀₀-maleimide, or maleimide-PEG_n-maleimide (wherein 1<n<5000) maleimide-OH, maleimide-PEG_n-OH (wherein 1)<n<5000) Maleimide-poly (ethylene glycol) -b-poly (. epsilon. -caprolactone), (S) - (-) -N- (1-phenylethyl) maleimide, N- (4-chlorophenyl) maleimide, N- (1-pyrenyl) maleimide, methoxypolyethylene glycol maleimide, poly (ethylene glycol) methyl ether maleimide, N- (4,4,5,5,6,6,7,7,8,8,9,9,10,10,11, 11-heptadecafluoroundecyl) maleimide, desferrioxamine-maleimide (i.e., chelator-maleimide), maleimide glycidyl ether, bifunctional maleimide DTPA, bifunctional NOTA-maleimide chelator, homobifunctional maleimide crosslinker (i.e., those having a maleimide group at each terminus), bismaleimide polyalkylene glycols, DBCO-maleimides, benzotriazole maleimides, alkyne maleimides, maleimide functionalized lipids, maleimide functionalized PEG lipids, and any molecule that typically comprises at least one maleimide moiety, at least one additional reactive moiety (e.g., maleimide or another reactive group), and one or more optional linkers (e.g., PEG or another polymer, such as polyglycerol).

II.B. joint

An EV (e.g., exosome) of the present disclosure may comprise one or more linkers that link (i.e., link) the maleimide moiety to a bioactive molecule or EV (e.g., exosome). In some aspects, the maleimide moiety is attached to the biologically active molecule via a linker. The linker can be any chemical moiety capable of, for example, attaching a maleimide moiety (e.g., formula (II) or (III)) to a biologically active molecule. In some aspects, the maleimide moiety may comprise one or more linkers. In some aspects, linkers disclosed herein or combinations thereof can be used to link, for example, a maleimide moiety to a bioactive molecule, a first bioactive moiety to a second bioactive moiety, an EV (e.g., a membrane lipid or scaffold protein thereof) to a maleimide moiety, or an EV (e.g., a membrane lipid or scaffold protein thereof) to a bioactive moiety.

In some aspects, the term "linker" refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) or a non-polypeptide, such as an alkyl chain. In some aspects, two or more linkers can be connected in series. When multiple linkers are present in the maleimide moiety disclosed herein, each linker may be the same or different. Generally, the joint provides flexibility or prevents/improves space obstruction. The joint is not typically cut; however, in certain aspects, such cutting may be desirable. Thus, in some aspects, the linker may comprise one or more protease cleavable sites, which may flank the linker within the linker sequence or at either end of the linker sequence.

In some aspects, the linker is a peptide linker. In some aspects, a peptide linker can comprise at least about two, at least about three, at least about four, at least about five, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 amino acids.

In some aspects, a peptide linker can comprise at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, or at least about 200 amino acids.

In other aspects, the peptide linker can comprise at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1,000 amino acids. The peptide linker may comprise 1 to about 5 amino acids, 1 to about 10 amino acids, 1 to about 20 amino acids, about 10 to about 50 amino acids, about 50 to about 100 amino acids, about 100 to about 200 amino acids, about 200 to about 300 amino acids, about 300 to about 400 amino acids, about 400 to about 500 amino acids, about 500 to about 600 amino acids, about 600 to about 700 amino acids, about 700 to about 800 amino acids, about 800 to about 900 amino acids, or about 900 to about 1000 amino acids.

In some aspects, the linker is a glycine/serine linker. In some aspects, the peptide linker is a glycine/serine linker according to the formula [ (Gly) n-Ser ] m (SEQ ID NO:46), wherein n is any integer from 1 to 100, and m is any integer from 1 to 100. In other aspects, the glycine/serine linker is a linker according to the formula [ (Gly) x-Sery ] z (SEQ ID NO:47), wherein x is an integer from 1 to 4, y is 0 or 1, and z is an integer from 1 to 50. In some aspects, the peptide linker comprises the sequence Gn (SEQ ID NO:48), wherein n can be an integer from 1 to 100. In some aspects, the peptide linker may comprise the sequence (GlyAla) n (SEQ ID NO:49), wherein n is an integer between 1 and 100. In other aspects, the peptide linker can comprise the sequence (GlyGlySer) n (SEQ ID NO:50), where n is an integer between 1 and 100.

In a particular aspect, the sequence of the peptide linker is GGGG (SEQ ID NO: 30).

In some aspects, the peptide linker may comprise the sequence (GlyAla) n, wherein n is an integer between 1 and 100. In other aspects, the peptide linker may comprise the sequence (GlyGlySer) n, wherein n is an integer between 1 and 100.

In other aspects, the peptide linker comprises the sequence (GGGS) n (SEQ ID NO: 31). In still other aspects, the peptide linker comprises the sequence (GGS) n (GGGGS) n (SEQ ID NO: 217). In these cases, n may be an integer from 1 to 100. In other cases, n may be an integer from one to 20, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some aspects, n is an integer from 1 to 100.

Additional examples of linkers include, but are not limited to, GGG, SGGSGGS (SEQ ID NO:218), GGSGGSGGSGGSGGG (SEQ ID NO:219), GGSGGSGGGGSGGGGS (SEQ ID NO:220), GGSGGSGGSGGSGGSGGS (SEQ ID NO:221), or GGGGSGGGGSGGS (SEQ ID NO: 222). In some aspects, the linker is a poly G sequence (GGGG) n (SEQ ID NO:223), where n can be an integer from 1 to 100.

In some aspects, the peptide linker is synthetic, i.e., not naturally occurring. In one aspect, a peptide linker comprises a peptide (or polypeptide) (e.g., a naturally or non-naturally occurring peptide) comprising a first linear amino acid sequence linked or genetically fused to a second linear amino acid sequence to which the first linear amino acid sequence is not naturally linked or genetically fused in nature. For example, in one aspect, a peptide linker can comprise a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide (e.g., comprising a mutation such as an addition, substitution, or deletion).

In other aspects, the peptide linker can comprise non-naturally occurring amino acids. In still other aspects, the peptide linker can comprise a naturally occurring amino acid that is present in a linear sequence that is not present in nature. In still others, the peptide linker may comprise a naturally occurring polypeptide sequence.

In some aspects, the linker comprises a non-peptide linker. In other aspects, the linker consists of a non-peptide linker. In some aspects, the non-peptide linker can be, for example, Maleimidocaproyl (MC), Maleimidopropanoyl (MP), methoxypolyethylene glycol (MPEG), 4- (N-maleimidomethyl) -cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4- (p-maleimidophenyl) butyric acid succinimidyl ester (SMPB), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), 6- [3- (2-pyridyldithio) -propionamide ] hexanoic acid succinimidyl ester (LC-SPDP), 4-succinimidoyloxycarbonyl- α -methyl- α - (2-pyridyldithio) toluene (SMPT), and the like (see, for example, U.S. patent No. 7,375,078).

Linkers can be introduced to the maleimide moiety using techniques known in the art (e.g., chemical conjugation, recombinant techniques, or peptide synthesis). In some aspects, linkers can be introduced using recombinant techniques. In other aspects, linkers can be introduced using solid phase peptide synthesis. In certain aspects, the maleimide moieties disclosed herein may contain both one or more linkers that have been introduced using recombinant techniques and one or more linkers that have been introduced using solid phase peptide synthesis or chemical conjugation methods known in the art.

The linker may be sensitive to cleavage ("cleavable linker") to facilitate release of the bioactive molecule. Thus, in some aspects, the maleimide moieties disclosed herein may comprise a cleavable linker. Such cleavable linkers may be susceptible to, for example, acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, under conditions in which the biologically active molecule remains active. Alternatively, the linker may be substantially resistant to cleavage ("non-cleavable linker").

Some cleavable linkers are cleaved by a protease ("protease-cleavable linkers"). Only certain peptides are readily cleaved intracellularly or extracellularly. See, for example, Trout et al, 79 Proc. Natl. Acad. Sci. USA,626-629(1982) and Umemoto et al, 43 int. J. cancer,677-684 (1989). The cleavable linker may comprise a cleavable site consisting of an alpha-amino acid unit and a peptide bond, which are chemically amide bonds between the carboxylic acid of one amino acid and the amino group of a second amino acid. Other amide bonds, such as the bond between the alpha-amino acid groups of carboxylic acids and lysine, are understood to be not peptide bonds and are considered non-cleavable.

In some aspects, the protease cleavable linker comprises a cleavage site for the protease: for example, neprilysin (CALLA or CDIO), phorbol oligopeptidase (TOP), leukotriene A4 hydrolase, endothelin converting enzyme, ste24 protease, lysin, mitochondrial intermediate peptidase, interstitial collagenase, stromelysin, macrophage elastase, stromelysin, gelatinase, transmembrane peptidase (meprins), procollagen C-endopeptidase, procollagen N-endopeptidase, ADAM and ADAMT metalloproteases, myelin-associated metalloproteases, amelysin, tumor necrosis factor alpha-converting enzyme, insulin lysin, nardilysin, mitochondrial processing peptidase, magnolysin, dactylysin-like metalloproteases, neutrophil collagenase, matrix metalloproteases, membrane-type matrix metalloproteases, SP2 endopeptidase, Prostate Specific Antigen (PSA), plasmin, urokinase, human fibroblast activation protein (FAP alpha), Trypsin, chymotrypsin, calpain (caldecrin), pancreatic elastase, pancreatic endopeptidase, enteropeptidase, leukocyte elastase, myeloblasts, chymotrypsin-like enzymes (chymas), tryptase, granzyme, cuticle chymotrypsin, acrosin, kallikrein, complement components and factors, alternative complement pathway c3/c5 convertase, mannose binding protein-associated serine proteases, coagulation factors, thrombin, protein c, u and t plasminogen activators, cathepsin G, hepatic serine (hepsin), serine proteolytic enzyme (prostasin), hepatocyte growth factor-activating endopeptidase, subtilisin/kexin type preprotein convertase, furin, proprotein convertase, prolyl peptidase, acylaminoacyl peptidase, peptidyl-glycaminase, signal peptidase, n-terminal nucleophile amino hydrolase, amino acid hydrolase, peptidyl-peptidase, peptidyl-glycanase, signal peptidase, and related enzymes, 20s proteasome, gamma-glutamyltranspeptidase, mitochondrial endopeptidase Ia, htra2 peptidase, proteolytic enzyme (matriptase), site 1 protease, asparaginyl endopeptidase (legumain), cathepsin, cysteine cathepsin, calpain, ubiquitin isopeptidase T, caspase, glycosylphosphatidylinositol protein transamidase, cancer procoagulant, prohormone thiol protease, gamma-glutamyl endopeptidase, bleomycin hydrolase, fibroblast activation protein (seprase), cathepsin B, cathepsin D, cathepsin L, cathepsin M, cathepsin K, pepsin (peps), chymosin (chymosyn), pepsin (gasstricin), renin, yeast aspartase (yapsin) and/or memapsin, Prostate Specific Antigen (PSA) or in general any Asp-N, Glu-C, Lys-C or Arg-C protease. See, for example, Cancer Res.77(24): 7027-. In some aspects, a cleavable linker component includes a peptide comprising one to ten amino acid residues. In these aspects, the peptides allow cleavage of the linker by proteases, thereby promoting release of the bioactive molecule upon exposure to intracellular proteases, such as lysosomal enzymes (Doronina et al (2003) nat. Biotechnol.21: 778-784). Exemplary peptides include, but are not limited to, dipeptides, tripeptides, tetrapeptides, pentapeptides, and hexapeptides. Exemplary dipeptides include, but are not limited to, valine-alanine (val-ala), valine-citrulline (val-cit), phenylalanine-lysine (phe-lys), N-methyl-valine-citrulline, cyclohexylalanine-lysine, and β -alanine-lysine. Exemplary tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine (gly-gly-gly).

The peptide may comprise naturally occurring and/or non-natural amino acid residues. The term "naturally occurring amino acid" refers to Ala, Asp, Cys, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr. "unnatural amino acids" (i.e., amino acids that are not naturally occurring) include, by way of non-limiting example, homoserine, homoarginine, citrulline, phenylglycine, taurine, iodotyrosine, selenocysteine, norleucine ("Nle"), norvaline ("Nva"), β -alanine, L-or D-naphthylamine, ornithine ("Orn"), and the like. The peptides can be designed and optimized for enzymatic cleavage by specific enzymes, for example, tumor-associated proteases, cathepsin B, C and D, or plasmin protease.

Amino acids also include the D forms of natural and unnatural amino acids. "D-" means an amino acid having the "D" (dextrorotatory) configuration, as opposed to the configuration found in naturally occurring ("L-") amino acids. Natural and unnatural amino acids are commercially available (Sigma Chemical co., Advanced Chemtech) or synthesized using methods known in the art.

Some linkers are cleaved by esterases ("esterase cleavable linkers"). Only certain esters can be cleaved by esterases present inside and outside the cell. Esters are formed by the condensation of carboxylic acids and alcohols. Simple esters are esters produced with simple alcohols (such as aliphatic alcohols) and small cyclic and aromatic alcohols.

In some aspects, the linker is a "reduction-sensitive linker". In some aspects, the reduction-sensitive linker comprises a disulfide bond. In some aspects, the linker is an "acid labile linker". In some aspects, the acid labile linker contains a hydrazone. Suitable acid-labile linkers also include, for example, cis-aconitic acid linkers, hydrazide linkers, thiocarbamoyl linkers, or any combination thereof.

In some aspects, the linker comprises a non-cleavable linker. A non-cleavable linker is any chemical moiety capable of linking a maleimide moiety to a biologically active molecule in a stable covalent manner and does not belong to the class of cleavable linkers listed above. Thus, non-cleavable linkers are substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. Further, non-cleavable refers to the ability of a chemical bond in or adjacent to a linker to undergo cleavage induced by an acid, a photolabile cleavage agent, a peptidase, an esterase, or a chemical or biochemical compound that cleaves a disulfide bond, without the cyclic dinucleotide and/or the antibody losing its activity. In some aspects, the bioactive molecule is linked to the linker via a spacer. In one aspect, the spacer is a self-immolative spacer. In another aspect, the spacer is a non-self-immolative spacer.

In some aspects, the linker comprises a non-cleavable linker comprising, for example, tetraethylene glycol (TEG), hexaethylene glycol (HEG), polyethylene glycol (PEG), succinimide, or any combination thereof. In some aspects, the non-cleavable linker comprises a spacer unit that links the biologically active molecule to the non-cleavable linker. In some aspects, one or more non-cleavable linkers comprise smaller units (e.g., HEG, TEG, glycerol, C2-C12 alkyl, etc.) linked together. In one aspect, the bond is an ester bond (e.g., a phosphodiester or phosphorothioate) or other bond.

II.B.1 ethylene glycol (HEG, TEG, PEG) linker

In some aspects, the linker comprises a non-cleavable linker, wherein the non-cleavable linker comprises polyethylene glycol (PEG), characterized by formula R³-(O-CH₂-CH₂)_n-or R³-(0-CH₂-CH₂)_n-O-wherein R³Is hydrogen, methyl or ethyl and n has a value of from 2 to 200. In some aspectsThe linker comprises a spacer, wherein the spacer is PEG.

In some aspects, the PEG linker is an oligo-ethylene glycol, such as a diethylene glycol, triethylene glycol, tetraethylene glycol (TEG), pentaethylene glycol, or hexaethylene glycol (HEG) linker.

In some aspects, n has a value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 110, 108, 111, 112, 125, 111, 122, 114, 122, 116, 114, 121, 122, 116, 121, 114, 122, 116, 121, 122, 121, 112, 122, 112, 121, 122, 112, 122, 112, 121, 112, 113, 112, 113, 121, 113, 112, 113, 116, 113, 116, 113, 112, 113, 116, 113, 112, 113, 21, 23, 21, 23, 60, 23, 60, 23, 21, 23, 61, 60, 21, 60, 23, 21, 60, 61, 23, 60, 61, and so, 126. 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 189, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200.

In some aspects, n is between 2 and 10, between 10 and 20, between 20 and 30, between 30 and 40, between 40 and 50, between 50 and 60, between 60 and 70, between 70 and 80, between 80 and 90, between 90 and 100, between 100 and 110, between 110 and 120, between 120 and 130, between 130 and 140, between 140 and 150, between 150 and 160, between 160 and 170, between 170 and 180, between 180 and 190, or between 190 and 200.

In some particular aspects, n has a value of 3 to 200, 3 to 20, 10 to 30, or 9 to 45.

In some aspects, the PEG is a branched PEG. Branched PEG has three to ten PEG chains emanating from a central core group.

In certain aspects, the PEG moiety is a monodisperse polyethylene glycol. In the context of the present disclosure, monodisperse polyethylene glycol (mdPEG) is PEG having a single, defined chain length and molecular weight. mdPEG is typically isolated from the polymerization mixture by chromatography. In certain formulae, monodisperse PEG moieties are designated by the abbreviation mdPEG.

In some aspects, the PEG is a star PEG. Star-shaped PEG has 10 to 100 PEG chains emanating from one central core group.

In some aspects, the PEG is comb PEG. Comb-shaped PEGs have multiple PEG chains typically grafted to a polymer backbone.

In certain aspects, the molar mass of PEG is between 100g/mol and 3000g/mol, specifically between 100g/mol and 2500g/mol, more specifically about 100g/mol to 2000 g/mol. In certain aspects, the molar mass of the PEG is between 200g/mol and 3000g/mol, specifically between 300g/mol and 2500g/mol, more specifically about 400g/mol to 2000 g/mol.

In some aspects, the PEG is PEG₁₀₀、PEG₂₀₀、PEG₃₀₀、PEG₄₀₀、PEG₅₀₀、PEG₆₀₀、PEG_700、PEG₈₀₀、PEG₉₀₀、PEG₁₀₀₀、PEG₁₁₀₀、PEG₁₂₀₀、PEG₁₃₀₀、PEG₁₄₀₀、PEG₁₅₀₀、PEG₁₆₀₀、PEG₁₇₀₀、PEG₁₈₀₀、PEG₁₉₀₀、PEG₂₀₀₀、PEG₂₁₀₀、PEG₂₂₀₀、PEG₂₃₀₀、PEG₂₄₀₀、PEG₂₅₀₀、PEG₁₆₀₀、PEG₁₇₀₀、PEG₁₈₀₀、PEG₁₉₀₀、PEG₂₀₀₀、PEG₂₁₀₀、PEG₂₂₀₀、PEG₂₃₀₀、PEG₂₄₀₀、PEG₂₅₀₀、PEG₂₆₀₀、PEG₂₇₀₀、PEG₂₈₀₀、PEG_{2900 or}PEG₃₀₀₀. In a particular aspect, the PEG is PEG₄₀₀. In another particular aspect, the PEG is PEG₂₀₀₀。

In some aspects, a linker of the present disclosure can comprise several PEG linkers, e.g., cleavable linkers flanking a PEG, HEG, or TEG linker.

In some aspects, the linker comprises (HEG) n and/or (TEG) n, where n is an integer between 1 and 50, and each unit is linked, e.g., via a phosphate linker, a phosphorothioate linkage, or a combination thereof.

II.B.2 Glycerol and Polyglycerol (PG)

In some aspects, the linker comprises a non-cleavable linker comprising a glycerol unit or consisting of the formula ((R)₃-O-(CH₂-CHOH-CH₂O)_n-) wherein R3 is hydrogen, methyl or ethyl, and n has a value of from 3 to 200. In some aspects, n has a value of 3 to 20. In some aspects, n has a value of 10 to 30.

In some aspects, the PG linker is a diglycerol, triglycerol, Tetraglycerol (TG), pentaglycerol, or Hexaglycerol (HG) linker.

In some alternatives of these aspects, n has a value of 9 to 45. In some aspects, the heterologous moiety is represented by the formula (R)³-O-(CH₂-CHOR⁵-CH₂-O)_n-) of branched polyglycerols described in which R is⁵Is hydrogen or of the formula (R)³-O-(CH₂-CHOH-CH₂-O)_n-) linear glycerol chain as described, and R³Is hydrogen, methyl or ethyl. In some aspects, the heterologous moiety is represented by the formula (R)³-O-(CH₂-CHOR⁵-CH₂-O)_n-) described in (a) a hyperbranched polyglycerol wherein R is⁵Is hydrogen or of the formula (R)³-O-(CH₂-CHOR⁶-CH₂-O)_n-) of the formula (I), wherein R is⁶Is hydrogen or of the formula (R)³-O-(CH₂-CHOR⁷-CH₂-O)_n-) of the formula (I), wherein R is⁷Is hydrogen or of the formula (R)³-O-(CH₂-CHOH-CH₂-O)_n-) linear glycerol chain described, and R³Is hydrogen, methyl or ethyl. Hyperbranched glycerol and a synthetic method thereof are disclosed in Oudshorn et al (2006) Biomaterials 27: 5471-5479; wilms et al (20100Acc. chem. Res.43,129-41 and references cited herein).

In certain aspects, the molar mass of PG is between 100g/mol and 3000g/mol, specifically between 100g/mol and 2500g/mol, more specifically about 100g/mol to 2000 g/mol. In certain aspects, the molar mass of PG is between 200g/mol and 3000g/mol, specifically between 300g/mol and 2500g/mol, more specifically about 400g/mol to 2000 g/mol.

In some aspects, PG is PG₁₀₀、PG₂₀₀、PG₃₀₀、PG₄₀₀、PG₅₀₀、PG₆₀₀、PG₇₀₀、PG₈₀₀、PG₉₀₀、PG₁₀₀₀、PG₁₁₀₀、PG₁₂₀₀、PG₁₃₀₀、PG₁₄₀₀、PG₁₅₀₀、PG₁₆₀₀、PG₁₇₀₀、PG₁₈₀₀、PG₁₉₀₀、PG₂₀₀₀、PG₂₁₀₀、PG₂₂₀₀、PG₂₃₀₀、PG₂₄₀₀、PG₂₅₀₀、PG₁₆₀₀、PG₁₇₀₀、PG₁₈₀₀、PG₁₉₀₀、PG₂₀₀₀、PG₂₁₀₀、PG₂₂₀₀、PG₂₃₀₀、PG₂₄₀₀、PG₂₅₀₀、PG₂₆₀₀、PG₂₇₀₀、PG₂₈₀₀、PG₂₉₀₀Or PG₃₀₀₀. In a particular aspect, PG is PG₄₀₀. In another particular aspect, PG is PG₂₀₀₀。

In some aspects, the linker comprises (glycerol) n, and/or (HG) n and/or (TG) n, wherein n is an integer between 1 and 50, and each unit is connected, e.g., via a phosphate linker, a phosphorothioate linkage, or a combination thereof.

II.B.3 aliphatic (alkyl) linkers

In some aspects, the linker comprises at least one aliphatic (alkyl) linker, for example, propyl, butyl, hexyl, or C2-C12 alkyl (such as C2-C10 alkyl or C2-C6 alkyl).

In some aspects, the linker comprises an alkyl chain, e.g., an unsubstituted alkyl group. In some aspects, a linker combination comprises a substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocycloalkylalkyl, heterocycloalkylalkenyl, heterocycloalkylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, heteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkynyl, and heteroaryl alkynyl, An alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclenyl, or alkenylheterocyclylalkynyl.

Optionally, these components are substituted. Substituents include alcohols, alkoxy groups (such as methoxy, ethoxy and propoxy), straight or branched alkyl groups (such as C1-C12 alkyl), amines, aminoalkyl groups (such as amino C1-C12 alkyl), phosphoramidites, phosphates, phosphoramidates, dithiophosphates, thiophosphates, hydrazides, hydrazines, halogens (such as F, Cl, Br or I), amides, alkylamides (such as amide C1-C12 alkyl), carboxylic acids, carboxylic esters, carboxylic anhydrides, carboxylic halides, ethers, sulfonyl halides, imidoesters, isocyanates, isothiocyanates, haloformates, carbodiimide adducts, aldehydes, ketones, sulfhydryls, haloacetyl, haloalkanes, alkylsulfonates, C (═ O) CH ═ CHC (═ O) (maleimide), thioethers, cyano groups, sugars (such as mannose, galactose and glucose), α, β -unsaturated carbonyl groups, Alkyl mercury or alpha, beta-unsaturated sulfone.

Unless otherwise specified, the term "alkyl" by itself or as part of another substituent means a straight or branched chain hydrocarbon radical (e.g., C) having the indicated number of carbon atoms₁-C₁₀Meaning one to ten carbon atoms). Typically, the alkyl group will have from 1 to 24 carbon atoms, for example from 1 to 10 carbon atoms, from 1 to 8 carbon atoms, or from 1 to 6 carbon atoms. "lower alkyl" is an alkyl group having 1 to 4 carbon atoms. The term "alkyl" includes divalent and polyvalent groups. For example, where appropriate, the term "alkyl" includes "alkylene" when the formula indicates that the alkyl group is divalent or when the substituents are joined together to form a ring, for example. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, and homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, and n-octyl.

The term "alkylene" by itself or as part of another substituent refers to a divalent (diradical) alkyl group, wherein alkyl is as defined herein. "alkylene" is for example but not limited to-CH₂CH₂CH₂CH₂-. Typically, an "alkylene" group will have 1 to 24 carbon atoms, for example 10 or fewer carbon atoms (e.g., 1 to 8 or 1 to 6 carbon atoms). "lower alkylene" is an alkylene having 1 to 4 carbon atoms.

The term "alkenyl", by itself or as part of another substituent, refers to a straight or branched chain hydrocarbon group having 2 to 24 carbon atoms and at least one double bond. Typical alkenyl groups have 2 to 10 carbon atoms and at least one double bond. In one aspect, the alkenyl group has 2 to 8 carbon atoms or 2 to 6 carbon atoms and 1 to 3 double bonds. Exemplary alkenyl groups include ethenyl, 2-propenyl, 1-but-3-enyl, crotyl, 2- (butadienyl), 2, 4-pentadienyl, 3- (1, 4-pentadienyl), 2-isopentenyl, 1-pent-3-enyl, 1-hex-5-enyl, and the like.

The term "alkynyl", by itself or as part of another substituent, refers to a straight or branched chain, unsaturated or polyunsaturated hydrocarbon radical having 2 to 24 carbon atoms and at least one triple bond. Typical "alkynyl groups" have 2 to 10 carbon atoms and at least one triple bond. In one aspect of the invention, the alkynyl group has 2 to 6 carbon atoms and at least one triple bond. Exemplary alkynyl groups include prop-1-ynyl, prop-2-ynyl (i.e., propargyl), ethynyl, and 3-butynyl.

The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense and refer to an alkyl group attached to the remainder of the molecule through an oxygen atom, an amino group, or a sulfur atom, respectively.

The term "heteroalkyl," by itself or in combination with another term, is intended to mean a radical consisting of the indicated number of carbon atoms (e.g., C)₂-C₁₀Or C₂-C₈) And at least one heteroatom selected from, for example, N, O, S, Si, B, and P (in one embodiment, N, O and S), wherein the nitrogen, sulfur, and phosphorus atoms are optionally oxidized, and the nitrogen atom (S) are optionally quaternized. The heteroatom(s) is placed at any internal position of the heteroalkyl group. Examples of heteroalkyl groups include, but are not limited to, -CH₂-CH₂-O-CH₃、-CH₂-CH₂-NH-CH₃、-CH₂-CH₂-N(CH₃)-CH₃、-CH₂-S-CH₂-CH₃、-CH₂-CH₂-S(O)-CH₃、-CH₂-CH₂-S(O)₂-CH₃、-CH＝CH-O-CH₃、-CH₂-Si(CH₃)₃、-CH₂-CH＝N-OCH₃and-CH ═ CH-N (CH)₃)-CH₃. Up to two heteroatoms may be consecutive, such as, for example, -CH₂-NH-OCH₃and-CH₂-O-Si(CH₃)₃。

Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from a heteroalkyl radical, such as, but not limited to, -CH₂-CH₂-S-CH₂-CH₂-and-CH₂-S-CH₂-CH₂-NH-CH₂-. Typically, the heteroalkyl group will have from 3 to 24 atoms (carbon and heteroatoms, excluding hydrogen) (3-to 24-membered heteroalkyl groups). In another example, the heteroalkyl group has a total of 3 to 10 atoms (3-to 10-membered heteroalkyl group) or 3 to 8 atoms (3-to 8-membered heteroalkyl group). Where appropriate, the term "heteroalkyl" includes "heteroalkylene", for example, when the formula indicates that the heteroalkyl is divalent or when the substituents are joined together to form a ring.

The term "cycloalkyl" by itself or in combination with other terms means having from 3 to 24 carbon atoms, for example, from 3 to 12 carbon atoms (e.g., C)₃-C₈Cycloalkyl or C₃-C₆Cycloalkyl groups). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. The term "cycloalkyl" also includes bridged, polycyclic (e.g., bicyclic) structures such as norbornyl, adamantyl, and bicyclo [2.2.1]A heptyl group. "cycloalkyl" may be fused to at least one (e.g., 1 to 3) additional ring selected from aryl (e.g., phenyl), heteroaryl (e.g., pyridyl), and non-aromatic (e.g., carbocyclic or heterocyclic) rings. When "cycloalkyl" includes fused aryl, heteroarylOr heterocyclic, then the "cycloalkyl" is connected to the remainder of the molecule through a carbocyclic ring.

The terms "heterocyclic hydrocarbon", "heterocyclic", "heterocycle" or "heterocyclyl", by themselves or in combination with other terms, denote carbocyclic non-aromatic rings (e.g., 3-to 8-membered rings and, for example, 4-, 5-, 6-or 7-membered rings) containing at least one and up to 5 heteroatoms selected from, for example, N, O, S, Si, B and P (e.g., N, O and S), wherein the nitrogen, sulfur and phosphorus atoms are optionally oxidized, and the nitrogen atom (S) are optionally quaternized (e.g., 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur), or a fused ring system of 4-to 8-membered rings containing at least one and up to 10 heteroatoms (e.g., 1 to 5 heteroatoms selected from N, O and S), in stable combinations known to those skilled in the art. Exemplary heterocyclic hydrocarbon groups include fused phenyl rings. When a "heterocyclic" group includes a fused aryl, heteroaryl or cycloalkyl ring, then the "heterocyclic" group is attached to the remainder of the molecule through a heterocyclic ring. The heteroatom may occupy the position where the heterocycle is attached to the remainder of the molecule.

Exemplary heterocycloalkyl or heterocyclyl groups of the present invention include morpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl S, S-dioxide, piperazinyl, homopiperazinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, tetrahydropyranyl, piperidinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, homopiperidinyl, homomorpholinyl, homothiomorpholinyl (homothiomorpholinyl), homothiomorpholinyl S, S-dioxide, oxazolidinonyl, dihydropyrazolyl, dihydropyrrolyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidyl, dihydrofuranyl, dihydropyranyl, tetrahydrothienyl S-oxide, tetrahydrothienyl S, S-dioxide, homothiomorpholinyl S-oxide, 1- (1,2,5, 6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidyl, 3-piperidyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.

"aryl" means a 5-, 6-, or 7-membered aromatic carbocyclic group having a single ring (e.g., phenyl) or fused to other aromatic or non-aromatic rings (e.g., 1 to 3 other rings). When "aryl" includes a non-aromatic ring (such as in 1,2,3, 4-tetrahydronaphthyl) or heteroaryl, then the "aryl" is attached to the remainder of the molecule through an aromatic ring (e.g., a benzene ring). Aryl is optionally substituted (e.g., with 1 to 5 substituents described herein). In one example, the aryl group has 6 to 10 carbon atoms. Non-limiting examples of aryl groups include phenyl, 1-naphthyl, 2-naphthyl, quinoline, indanyl, indenyl, dihydronaphthyl, fluorenyl, tetrahydronaphthyl, benzo [ d ] [1,3] dioxolyl, or 6,7,8, 9-tetrahydro-5H-benzo [ a ] cycloheptenyl. In one aspect, the aryl group is selected from phenyl, benzo [ d ] [1,3] dioxolyl, and naphthyl. In yet another aspect, aryl is phenyl.

The term "arylalkyl" or "arylalkyl" is intended to include those groups in which an aryl or heteroaryl group is linked to an alkyl group to form the groups-alkyl-aryl and-alkyl-heteroaryl (wherein alkyl, aryl and heteroaryl are as defined herein). Exemplary "arylalkyl" or "aralkyl" groups include benzyl, phenethyl, pyridylmethyl and the like.

"aryloxy" means the group-O-aryl, wherein aryl is as defined herein. In one example, the aryl portion of the aryloxy group is phenyl or naphthyl. In one aspect, the aryl moiety of the aryloxy group is phenyl.

The term "heteroaryl" or "heteroaromatic" refers to a polyunsaturated 5-, 6-, or 7-membered aromatic moiety containing at least one heteroatom (e.g., 1 to 5 heteroatoms, such as 1-3 heteroatoms) selected from N, O, S, Si and B (e.g., N, O and S), wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom (S) are optionally quaternized. "heteroaryl" can be monocyclic or fused to other aryl, heteroaryl, cycloalkyl or heterocycloalkyl rings (e.g., 1 to 3 other rings). When "heteroaryl" includes a fused aryl, cycloalkyl or heterocycloalkyl ring, then "heteroaryl" is attached to the remainder of the molecule through a heteroaromatic ring. Heteroaryl groups may be attached to the rest of the molecule through a carbon or heteroatom.

In one example, the heteroaryl group has 4 to 10 carbon atoms and 1 to 5 heteroatoms selected from O, S and N. Non-limiting examples of heteroaryl groups include pyridyl, pyrimidinyl, quinolinyl, benzothienyl, indolyl, indolinyl, pyridazinyl, pyrazinyl, isoindolyl, isoquinolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, imidazolyl, isoxazolyl, pyrazolyl, oxazolyl, thiazolyl, indolizinyl, indazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, furyl, thienyl, pyrrolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, isothiazolyl, naphthyridinyl, isochromanyl, chromanyl, tetrahydroisoquinolinyl, isoindolinyl, isobenzotetrahydrofuryl, isobenzotetrahydrothienyl, isobenzothienyl, benzoxazolyl, pyridopyridyl, benzotetrahydrofuranyl, benzotetrahydrothienyl, purinyl, benzodioxolyl (benzodioxolyl), triazinyl, quinoxalinyl, and quinoxalinyl, Pteridinyl, benzothiazolyl, imidazopyridinyl, imidazothiazolyl, dihydrobenzisoxazinyl, benzisoxazinyl, benzoxazinyl, dihydrobenzisothiazinyl, benzopyranyl, benzothiopyranyl, chromonyl, chromanone, pyridyl-N-oxide, tetrahydroquinolinyl, dihydroquinolinyl, dihydroisoquinolinonyl, dihydrocoumarinyl, dihydroisocoumarinyl, isoindolinonyl, benzodioxanyl, benzoxazolinonyl, pyrrolyl N-oxide, pyrimidinyl N-oxide, pyridazinyl N-oxide, pyrazinyl N-oxide, quinolinyl N-oxide, indolyl N-oxide, indolinyl N-oxide, chromanyl, tetrahydroquinolinyl, dihydroquinolinyl, dihydroisoquinolinyl, benzodioxanyl, benzoxazolinonyl, pyrrolyl N-oxide, pyrimidinyl N-oxide, pyridazinyl N-oxide, pyrazinyl N-oxide, quinolinyl-N-oxide, indolyl N-oxide, indolinyl N-oxide, Isoquinolinyl N-oxide, quinazolinyl N-oxide, quinoxalinyl N-oxide, phthalazinyl N-oxide, imidazolyl N-oxide, isoxazolyl N-oxide, oxazolyl N-oxide, thiazolyl N-oxide, indolizinyl N-oxide, indazolyl N-oxide, benzothiazolyl N-oxide, benzimidazolyl N-oxide, pyrrolyl N-oxide, oxadiazolyl N-oxide, thiadiazolyl N-oxide, triazolyl N-oxide, tetrazolyl N-oxide, benzothiopyranyl S, S-dioxide. Exemplary heteroaryl groups include imidazolyl, pyrazolyl, thiadiazolyl, triazolyl, isoxazolyl, isothiazolyl, imidazolyl, thiazolyl, oxadiazolyl, and pyridyl. Other exemplary heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, pyridin-4-yl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, 6-quinolyl. The substituents for each of the above-mentioned aromatic and heteroaromatic ring systems are selected from the group consisting of the acceptable aryl group substituents described below.

Examples of aliphatic linkers include the following structures: -O-CO-O-, -NH-CO-NH-, -NH (CH)₂)_n1—、—S—(CH₂)_n1—、—CO—(CH₂)_n1—CO—、—CO—(CH₂)_n1—NH—、—NH—(CH₂)_n1—NH—、—CO—NH—(CH₂)_n1—NH—CO—、—C(═S)—NH—(CH₂)_n1—NH—CO—、—C(═S)—NH—(CH₂)_n1—NH—C—(═S)—、—CO—O—(CH₂)_n1—O—CO—、—C(═S)—O—(CH₂)_n1—O—CO—、—C(═S)—O—(CH₂)_n1—O—C—(═S)—、—CO—NH—(CH₂)_n1—O—CO—、—C(═S)—NH—(CH₂)_n1—O—CO—、—C(═S)—NH—(CH₂)_n1—O—C—(═S)—、—CO—NH—(CH₂)_n1—O—CO—、—C(═S)—NH—(CH₂)_n1—CO—、—C(═S)—O—(CH₂)_n1—NH—CO—、—C(═S)—NH—(CH₂)_n1—O—C—(═S)—、—NH—(CH₂CH₂O)_n2—CH(CH₂OH)—、—NH—(CH₂CH₂O)_n2—CH₂—、—NH—(CH₂CH₂O)_n2—CH₂—CO—、—O—(CH₂)_n3—S—S—(CH₂)_n4—O—P(═O)₂—、—CO—(CH₂)_n3—O—CO—NH—(CH₂)_n4—、—CO—(CH₂)_n3—CO—NH—(CH₂)_n4—、—(CH2)_n1NH—、—C(O)(CH2)_n1NH—、—C(O)—(CH2)_n1-C(O)—、—C(O)—(CH2)_n1-C(O)O—、—C(O)—O—、—C(O)—(CH2)_n1-NH—C(O)—、—C(O)—(CH2)_n1—、—C(O)—NH—、—C(O)—、—(CH2)_n1-C(O)—、—(CH2)_n1-C(O)O—、—(CH2)_n1—、—(CH2)_n1-NH — c (o) -, wherein n1 is an integer between 1 and 40 (e.g., 2 to 20, or 2 to 12); n2 is an integer between 1 and 20 (e.g., 1 to 10, or 1 to 6); n3 and n4 may be the same or different and are integers between 1 and 20 (e.g., 1 to 10 or 1 to 6).

In some aspects, the linker comprises (C3) n, (C4) n, (C5) n, (C6) n, (C7) n, or (C8) n, or a combination thereof, wherein n is an integer between 1 and 50, and each unit is linked, e.g., via a phosphate linker, a phosphorothioate linkage, or a combination thereof.

II.B.4 cleavable linkers

In some aspects, the linker may be a cleavable linker. The term cleavable linker refers to a linker comprising at least one cleavable or cleavable bond or chemical bond. As used herein, the term cleavage refers to the disruption of one or more chemical bonds in a relatively large molecule in a manner that produces two or more relatively small molecules. Cleavage may be mediated by, for example, nucleases, peptidases, proteases, phosphatases, oxidases or reductases, for example, or by specific physicochemical conditions, such as redox environment, pH, presence of reactive oxygen species or light of a specific wavelength.

In some aspects, as used herein, the term "cleavable" refers to, for example, rapidly degradable linkers, e.g., phosphodiesters and disulfide bonds, while the term "non-cleavable" refers to, for example, more stable linkages, e.g., nuclease-resistant phosphorothioates. In some aspects, the cleavable linker is a di-or trinucleotide linker, a disulfide, an imine, a thioketal, a val-cit dipeptide, or any combination thereof. In some aspects, the cleavable linker comprises a valine-alanine-p-aminobenzyl carbamate or a valine-citrulline-p-aminobenzyl carbamate.

II.B.4.a Redox cleavable linker

In some aspects, the linker comprises a redox cleavable linker. As a non-limiting example, one type of cleavable linker is a redox-cleavable linking group that is cleaved upon reduction or oxidation. In some aspects, the redox-cleavable linker comprises a disulfide bond, i.e., it is a disulfide-cleavable linker. The redox-cleavable linker may be reduced, for example, by an intracellular thiol, oxidase or reductase.

II.B.4.b Reactive Oxygen Species (ROS) -cleavable linkers

In some aspects, the linker can comprise a cleavable linker that can be cleaved by a Reactive Oxygen Species (ROS), such as superoxide (O)₂-) or hydrogen peroxide (H)²O₂) Cutting machine_CuttingE.g. produced by inflammatory processes such as activated neutrophils. In some aspects, the ROS-cleavable linker is a thioketal cleavable linker. See, for example, U.S. patent 8,354,455B2, which is incorporated by reference herein in its entirety.

II.B.4.c pH-dependent cleavable linkers

In some aspects, the linker is an "acid labile linker" comprising an acid cleavable linking group that is a linking group that selectively cleaves under acidic conditions (pH < 7). As a non-limiting example, the acid-cleavable linking group is cleaved in an acidic environment, e.g., about 6.0, 5.5, 5.0 or less. In some aspects, the pH is about 6.5 or less. In some aspects, the linker is cleaved by an agent, such as an enzyme, which can act as a universal acid, e.g., a peptidase (which may be substrate specific) or phosphatase. Within the cell, certain low pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linking group. Although the pH of human serum is 7.4, the average pH inside the cells is slightly lower, about 7.1 to 7.3. Endosomes also have acidic pH values ranging from 5.5 to 6.0, while lysosomes are about 5.0 at more acidic pH values. Thus, the art sometimes refers to pH-dependent cleavable linkers as endosomal labile linkers.

The acid cleavable group may have the general formula-C ═ NN-, C (O) O, or-oc (O). In another non-limiting example, when a carbon is attached to an ester oxygen (alkoxy), it is attached to an aryl, substituted alkyl, or tertiary alkyl group, such as dimethylpentyl or tertiary butyl. Examples of acid-cleavable linkers include, but are not limited to, amines, imines, amino esters, benzimines, di-ortho esters, polyphosphates, polyphosphazenes, acetals, vinyl ethers, hydrazones, cis-aconitates, hydrazides, thiocarbamoyl, imines, azidomethyl-methylmaleic anhydride, thiopropionates, masked endosomolytic agents, citraconyl, or any combination thereof. Disulfide bonds are also susceptible to pH.

In some aspects, the linker comprises a low pH labile hydrazone bond. Such acid labile bonds have been widely used in the field of conjugates, such as antibody-drug conjugates. See, e.g., Zhou et al (2011) Biomacromolecules 12: 1460-7; yuan et al (2008) Acta Biomate.4: 1024-37; zhang et al (2008) Acta Biomate.6: 838-50; yang et al (2007) J.Pharmacol.Exp.Ther.321: 462-8; reddy et al (2006) Cancer Chemother. Pharmacol.58: 229-36; doronina et al (2003) Nature Biotechnol.21:778-84, all of which are incorporated herein by reference in their entirety.

In certain aspects, the linker comprises a low pH labile bond selected from: ketals that are unstable in acidic environments (e.g., pH less than 7, greater than about 4) to form diols and ketones; acetals that form diols and aldehydes are unstable in acidic environments (e.g., pH less than 7, greater than about 4); imines or iminium salts that are unstable in acidic environments (e.g., pH less than 7, greater than about 4) to form amines and aldehydes or ketones; silicon-oxygen-carbon bonds which are unstable under acidic conditions; a silicon-nitrogen (silazane) bond; silicon-carbon bonds (e.g., aryl silanes, vinyl silanes, and allyl silanes); maleate (amide bond synthesized from maleic anhydride derivative and amine); an ortho ester; hydrazone; activated carboxylic acid derivatives (e.g., esters, amides) designed to undergo acid-catalyzed hydrolysis; or a vinyl ether.

Additional examples can be found in U.S. patent nos. 9,790,494B2 and 8,137,695B2, the contents of which are incorporated by reference herein in their entirety.

II.B.4.d enzymatically cleavable linkers

In some aspects, the linker may comprise a linker that is cleavable by an intracellular or extracellular enzyme (e.g., protease, esterase, nuclease, amidase). The range of enzymes that can cleave a particular linker in a linker combination depends on the particular bond and chemical structure of the linker. Thus, a peptide linker can be cleaved by, for example, a peptidase, and a linker containing an ester bond can be cleaved by, for example, an esterase; linkers containing amide bonds can be cleaved by, for example, amidases; and so on.

II.B.4.e protease cleavable linkers

In some aspects, the linker comprises a protease cleavable linker, i.e., a linker that can be cleaved by an endogenous protease. Only certain peptides are readily cleaved intracellularly or extracellularly. See, for example, Trout et al (1982) Proc. Natl. Acad. Sci. USA 79:626-629 and Umemoto et al (1989) int. J. cancer 43: 677-684. The cleavable linker may comprise a cleavable site consisting of an alpha-amino acid unit and a peptide bond, which are chemically amide bonds between the carboxylic acid of one amino acid and the amino group of a second amino acid. Other amide bonds, such as the bond between the alpha-amino acid groups of carboxylic acids and lysine, are understood to be not peptide bonds and are considered non-cleavable.

In some aspects, the protease cleavable linker comprises a cleavage site for the protease: for example, neprilysin (CALLA or CDIO), phorbol oligopeptidase (TOP), leukotriene A4 hydrolase, endothelin converting enzyme, ste24 protease, lysin, mitochondrial intermediate peptidase, interstitial collagenase, stromelysin, macrophage elastase, stromelysin, gelatinase, transmembrane peptidase (meprins), procollagen C-endopeptidase, procollagen N-endopeptidase, ADAM and ADAMT metalloproteases, myelin-associated metalloproteases, amelysin, tumor necrosis factor alpha-converting enzyme, insulin lysin, nardilysin, mitochondrial processing peptidase, magnolysin, dactylysin-like metalloproteases, neutrophil collagenase, matrix metalloproteases, membrane-type matrix metalloproteases, SP2 endopeptidase, Prostate Specific Antigen (PSA), plasmin, urokinase, human fibroblast activation protein (FAP alpha), Trypsin, chymotrypsin, calpain (caldecrin), pancreatic elastase, pancreatic endopeptidase, enteropeptidase, leukocyte elastase, myeloblasts, chymotrypsin-like enzymes (chymas), tryptase, granzyme, cuticle chymotrypsin, acrosin, kallikrein, complement components and factors, alternative complement pathway c3/c5 convertase, mannose binding protein-associated serine proteases, coagulation factors, thrombin, protein c, u and t plasminogen activators, cathepsin G, hepatic serine (hepsin), serine proteolytic enzyme (prostasin), hepatocyte growth factor-activating endopeptidase, subtilisin/kexin type preprotein convertase, furin, proprotein convertase, prolyl peptidase, acylaminoacyl peptidase, peptidyl-glycaminase, signal peptidase, n-terminal nucleophile amino hydrolase, amino acid hydrolase, peptidyl-peptidase, peptidyl-glycanase, signal peptidase, and related enzymes, 20s proteasome, gamma-glutamyltranspeptidase, mitochondrial endopeptidase Ia, htra2 peptidase, proteolytic enzyme (matriptase), site 1 protease, asparaginyl endopeptidase (legumain), cathepsin, cysteine cathepsin, calpain, ubiquitin isopeptidase T, caspase, glycosylphosphatidylinositol protein transamidase, cancer procoagulant, prohormone thiol protease, gamma-glutamyl endopeptidase, bleomycin hydrolase, fibroblast activation protein (seprase), cathepsin B, cathepsin D, cathepsin L, cathepsin M, cathepsin K, pepsin (peps), chymosin (chymosyn), pepsin (gasserin), renin, yeast aspartase (yapsin) and/or memapsin, Prostate Specific Antigen (PSA) or in general any Asp-N, Glu-C, Lys-C or Arg-C protease. See, for example, Cancer Res.77(24): 7027-. In some aspects, a cleavable linker component includes a peptide comprising one to ten amino acid residues. In these aspects, the peptides allow cleavage of the linker by proteases, thereby promoting release of the bioactive molecule upon exposure to intracellular proteases, such as lysosomal enzymes (Doronina et al (2003) nat. Biotechnol.21: 778-784). Exemplary peptides include, but are not limited to, dipeptides, tripeptides, tetrapeptides, pentapeptides, and hexapeptides.

Exemplary dipeptides include, but are not limited to, valine-alanine, valine-citrulline, phenylalanine-lysine, N-methyl-valine-citrulline, cyclohexylalanine-lysine, and β -alanine-lysine. Exemplary tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine (gly-gly-gly).

II.B.4.f esterase cleavable linkers

Some linkers are cleaved by esterases ("esterase cleavable linkers"). Only certain esters can be cleaved by esterases and amidases present inside and outside the cell. Esters are formed by the condensation of carboxylic acids and alcohols. Simple esters are esters produced with simple alcohols (such as aliphatic alcohols) and small cyclic and aromatic alcohols. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. The ester-cleavable linking group has the general formula-C (O) O-or-OC (O) -.

II.B.4.g phosphatase cleavable linkers

In some aspects, the linker combination may include a phosphate-based cleavable linking group that is cleaved by an agent that degrades or hydrolyzes the phosphate group. Examples of the agent that cleaves an intracellular phosphate group are such as intracellular phosphatase. An example of a phosphate-based linking group is-O-P (O) (OR)_k)—O—、—O—P(S)(OR_k)—O—、—O—P(S)(SR_k)—O-、-S-P(O)(OR_k)-O-、-O-P(O)(OR_k)-S-、-S-P(O)(OR_k)-S-、-O-P(S)(OR_k)-S-、-SP(S)(OR_k)-O-、-OP(O)(R_k)-O-、-OP(S)(R_k)-O-、-SP(O)(R_k)-O-、-SP(S)(R_k)-O-、-SP(O)(R_k) -S-or-OP (S) (R)_k) -S-, wherein R_kIs NH₂、BH₃、CH₃、C_1-6Alkyl radical, C_6-10Aryl radical, C_1-6Alkoxy or C_6-10An aryloxy group. In some aspects, C_1-6Alkyl and C_6-10The aryl group is unsubstituted. Further non-limiting examples are-O-P (O) (OH) -O-, -O-P (S) (SH) -O-, -S-P (O) (OH) -O-, -O-P (O) (OH) -S-, -S-P (O) (OH) -S-, -O-P (S) (OH) -S-, -S-P (S) (OH) -O-, -O-P (O) (H) -O-, -O-P (S) (H) -O-, -S-P (O) -O-, -S- (H) -O-, -SP (S) (H) -O-, (H-), (H) SP, (O) S-, -OP (S), (H) -S-, or-O-P (O) (OH) -O-.

II.B.4.h photoactivated cleavable linkers

In some aspects, the combination comprises a photoactivated cleavable linker, such as a nitrobenzyl linker or a linker comprising a nitrobenzyl reactive group.

II.C. self-immolative spacer

In some aspects, the autolytic spacer in an EV (e.g., exosome) of the present disclosure undergoes 1,4 elimination after enzymatic cleavage of the protease cleavable linker. In some aspects, the autolytic spacer in an EV (e.g., exosome) of the present disclosure undergoes 1,6 elimination after enzymatic cleavage of the protease cleavable linker. In some aspects, the self-immolative spacer is, for example, p-aminobenzyl carbamate (PABC), p-aminobenzyl ether (PABE), p-aminobenzyl carbonate, or a combination thereof.

In certain aspects, the self-immolative spacer comprises an aryl group. In some aspects, aryl is selected from benzyl, cinnamyl, naphthyl, and biphenyl. In some aspects, the aryl is heterocyclic. In other aspects, the aryl group comprises at least one substituent. In some aspects, at least one substituent is selected from the group consisting of: F. cl, I, Br, OH, methyl, methoxy, NO₂、NH₂、NO³⁺、NHCOCH₃、N(CH₃)₂、NHCOCF₃Alkyl, haloalkyl, C₁-C₈Alkyl halides, carboxylates, sulfates, sulfamates, and sulfonates.

In other aspects, at least one C in the aromatic group is substituted with N, O or CR ", wherein R" is independently selected from H, F, Cl, I, Br, OH, methyl, methoxy, NO₂、NH₂、NO³⁺、NHCOCH₃、N(CH₃)₂、NHCOCF₃Alkyl, haloalkyl, C₁-C₈Alkyl halides, carboxylates, sulfates, sulfamates, and sulfonates.

In some aspects, the self-immolative spacer comprises a aminobenzyl carbamate group, an aminobenzyl ether group, or an aminobenzyl carbonate group. In one aspect, the self-immolative spacer is p-aminobenzyl carbamate (PABC). Benzyl P-carbamate (PABC) is the most efficient and widespread linker linkage for self-cleaving site-specific prodrug activation (see, e.g., Carl et al (1981) J. Med. chem.24: 479-480; WO 1981/001145; Rautio et al (2008) Nature Reviews Drug Discovery 7: 255-270; Simplicio et al (2008) Molecules 13:519-547, the entire contents of which are incorporated herein by reference in their entirety). PABC allows the release of any amine drugs, peptides and proteins upon cleavage by proteases and 1,6 spontaneous cleavage.

In some aspects, a self-immolative spacer links a biologically active molecule (e.g., an antibody) to a protease-cleavable substrate. In particular aspects, the PABC is attached to the N-terminus of a biologically active molecule (e.g., an antibody) from the carbamate group of the cleavage spacer, and the PABC is attached to the protease-cleavable substrate from the amino group of the cleavage group.

The aromatic ring of the aminobenzyl group may optionally be substituted with one or more (e.g., R) groups on the aromatic ring₁And/or R₂) A substituent that replaces a hydrogen attached to one of the four unsubstituted carbons forming the ring. The symbol "R" as used herein_x"(e.g., R)₁、R₂、R₃、R₄) Are general abbreviations that represent substituents as described herein.

The substituent may improve the self-decomposition ability of p-aminobenzyl. See, Hay et al (1999) J.chem Soc., Perkin Trans.1: 2759-2770; see also Sykes et al J. (2000) chem.Soc., Perkin Trans.1: 1601-1608.

Can be eliminated, for example, by 1,4 elimination, 1,6 elimination (e.g., PABC), 1,8 elimination (e.g., p-aminocinnamoyl alcohol), β -elimination, cyclization elimination (e.g., 4-aminobutanol ester and ethylenediamine), cyclization/lactonization, and the like. See, for example, Singh et al (2008) curr. Med. chem.15: 1802-.

In some aspects, the self-immolative spacer can comprise, for example, a cinnamyl group, a naphthyl group, or a biphenyl group (see, e.g., Blencowee et al (2011) Polymer. chem.2: 773-. In some aspects, the self-immolative spacer comprises a heterocyclic ring (see, e.g., U.S. patent nos. 7,375,078, 7,754,681). Many are based on homoaromatics (see, e.g., Carl et al (1981) J.Med.chem.24: 479; Senter et al (1990) J.org.chem.55: 2975; Taylor et al (1978) J.org.chem.43: 1197; Andriamenjanhary et al (1992) bioorg.Med.chem.Lett.2:1903) and coumarins (see, e.g., Weinstein et al (2010) chem.Commun.46:553), furans, thiophenes, thiazoles, oxazoles, isoxazoles, pyrroles, pyrazoles (see, e.g., Hay et al (2003) J.Med.chem.5546: 33), pyridines (see, e.g., Perry-Feigenbaum et al (2009) org.Biomol. chem.7:4825), imidazoles (see, e.g., Nailnbaunbaun et al (2009) org.7: 4825), and physiological groups are known in the art (see, e.g.g.g.73, Textil.g.g.3572, and Lertn.g.73, and elsewhere (see, et al, physiological groups known in the art, see, e.3, see, et al, a physiological, a. See also, U.S. Pat. Nos. 7,691,962, 7,091,186 and U.S. patent publication Nos. US2006/0269480, US2010/0092496, US2010/0145036, US2003/0130189 and US2005/0256030, the entire contents of which are incorporated by reference in their entirety.

In some aspects, the maleimide moieties disclosed herein comprise more than one self-immolative spacer in series, e.g., two or more PABC units. See, e.g., de Groot et al (2001) j. org. chem.66: 8815-8830. In some aspects, the maleimide moieties disclosed herein can comprise a self-immolative spacer (e.g., p-aminobenzyl alcohol or a hemithioamino derivative of p-carboxybenzaldehyde or glyoxylic acid) attached to a fluorescent probe (see, e.g., Meyer et al (2010) org.biomol.chem.8: 1777-1780).

When a substituent in a self-immolative linker is specified by its conventional formula written from left to right, it also includes chemically identical substituents that would result from writing the structure from right to left. For example, "-CH₂O- "is also intended to be known as" -OCH₂-”。

Self-decomposition as discussed above, e.g. R in p-aminobenzyl self-decomposing linker₁And/or the substituent groups in the R2 substituent may include, for example, alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, aryloxy, heteroaryl, and the like. When a compound of the present disclosure comprises more than one substituent, then each substituent is independently selected.

In some particular aspects, the linker is of formula (IV):

-A_a-Y_y- (IV)，

wherein each-A-is independently an amino acid unit, a is independently an integer from 1 to 12; -Y-is a spacer unit and Y is 0, 1 or 2. In some aspects, -A_a-is a dipeptide, tripeptide, tetrapeptide, pentapeptide or hexapeptide. In some aspects, -A_a-selected from the group consisting of: valine-alanine, valine-citrulline, phenylalanine-lysine, N-methylvaline-citrulline, cyclohexylalanine-lysine and β -alanine-lysine. In some aspects, -A_a-is valine-alanine or valine-citrulline. In some aspects, y is 1. In some aspects, -Y-is a self-immolative spacer.

In some aspects, the self-immolative spacer-Y_y-has formula (V):

wherein each R²Independently is C_1-8Alkyl, -O- (C)_1-8Alkyl), halogen, nitro or cyano; and m is an integer of 0 to 4. In some aspects, m is 0, 1, or 2. In some aspects, m is 0.

In some aspects, the cleavable linker of formula (IV) is valine-alanine-p-aminobenzyl carbamate or valine-citrulline-p-aminobenzyl carbamate. In some aspects, the spacer unit-Y-is a non-self-immolative spacer, e.g., -Gly-or-Gly-.

In some aspects, the linker is a non-cleavable linker including, for example, tetraethylene glycol (TEG), polyethylene glycol (PEG), succinimide, or any combination thereof. In some aspects, the non-cleavable linker comprises a spacer unit that links the biologically active molecule to the non-cleavable linker.

In some aspects, the present disclosure provides an EV (e.g., exosome) comprising a bioactive molecule and a cleavable linker, wherein the cleavable linker connects the EV (e.g., exosome) to the bioactive molecule, and the cleavable linker comprises valine-alanine-p-aminobenzyl carbamate or valine-citrulline-p-aminobenzyl carbamate. In some aspects, the EV (e.g., exosome) further comprises a maleimide moiety that links the EV (e.g., exosome) to the cleavable linker via a functional group present on the EV (e.g., exosome). Suitable maleimide moieties are those described above, for example, maleimide moieties of formulae (I), (II) and (III). In some aspects, the maleimide moiety is covalently attached to a functional group present on the EV (e.g., exosome), wherein the functional group is a thiol (thiol), wherein the thiol group is on a protein on the surface of the EV (e.g., exosome), such as the outer surface of the EV (e.g., exosome).

II.D bioactive molecules

In some aspects, the EVs (e.g., exosomes) disclosed herein are capable of delivering a payload (e.g., a bioactive molecule chemically linked to the EV (e.g., exosome) via a maleimide moiety) to a target. The payload is an agent that acts on a target (e.g., a target cell) that is in contact with the EV (e.g., an exosome). The contacting can occur in vitro or in vivo in a subject. Non-limiting examples of payloads that can be linked (e.g., chemically linked via a maleimide moiety) to EVs (e.g., exosomes) include agents such as nucleotides (e.g., nucleotides comprising a detectable moiety or toxin or that disrupt transcription), nucleic acids (e.g., DNA or mRNA molecules encoding a polypeptide such as an enzyme, or RNA molecules with regulatory functions such as miRNA, dsDNA, incrna, or siRNA), morpholines, amino acids (e.g., amino acids comprising a detectable moiety or that disrupt translation of a toxin), polypeptides (e.g., enzymes), lipids, carbohydrates, small molecules (e.g., small molecule drugs and toxins), antigens (e.g., vaccine antigens), adjuvants, or combinations thereof.

In some aspects, an EV (e.g., exosome) may comprise more than one payload, such as a first payload in a luminal solution of the EV (e.g., exosome) and a second payload connected to, for example, an outer surface of the EV (e.g., exosome) via a maleimide moiety. In some aspects, the payload comprises a small molecule. In some aspects, the payload comprises a peptide. In some aspects, the payload comprises an antigen, such as a vaccine antigen. In some aspects, the payload comprises a vaccine adjuvant.

II.D.1 Targeted antigens and payloads of vaccine antigens

In some aspects, the payload interacts with an antigen, such as a tumor antigen. In some aspects, the biological function of an antigen (e.g., a tumor antigen) is modulated by interaction with a payload (e.g., if the antigen is a receptor, the payload can be a receptor agonist or receptor antagonist). In other aspects, the payload comprises an antigen capable of inducing an immune response (i.e., a vaccine antigen). In some aspects, the payload can comprise an antigen capable of inducing an immune response (i.e., a vaccine antigen) and an adjuvant (i.e., a vaccine adjuvant). In some aspects, the vaccine antigen and vaccine adjuvant may be on the same EV (e.g., exosome). In other aspects, the vaccine and vaccine adjuvant may be in different EVs (e.g., exosomes).

Non-limiting examples of tumor antigens include: alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), Epithelial Tumor Antigen (ETA), mucin 1(MUC1), Tn-MUC1, mucin 16(MUC16), tyrosinase, melanoma-associated antigen (MAGE), tumor protein p53(p53), CD4, CD8, CD45, CD80, CD86, programmed death ligand 1(PD-L1), programmed death ligand 2(PD-L2), NY-ESO-1, PSMA, TAG-72, HER2, GD2, ET, EGFR, mesothelin, VEGFR, alpha folate receptor, CE7R, IL-3, cancer-testis antigen (CTA), MART-1gp100, TNF-related apoptosis-inducing ligands, or combinations thereof.

Payloads that interact with tumor antigens and, for example, modulate their biological function include, for example, antibodies and binding fragments thereof, aptamers, Antibody Drug Conjugates (ADCs), and small molecules.

In some aspects, the antigen is a universal tumor antigen. As used herein, the term "universal tumor antigen" refers to an immunogenic molecule, such as a protein, that is, is typically expressed at a higher level in tumor cells than in non-tumor cells, and is also expressed in tumors of different origin. In some aspects, the universal tumor antigen is expressed in more than about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the cancers (e.g., human cancers). In some aspects, a universal tumor antigen can be expressed in a non-tumor cell (e.g., a normal cell) but at a level that is lower than its expression level in a tumor cell. In certain aspects, the universal tumor antigen is expressed at a level on the tumor cell that is about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold or more compared to a non-tumor cell. In certain aspects, the universal tumor antigen is not expressed in normal cells but only in tumor cells. Non-limiting examples of universal tumor antigens that may be used with the present disclosure include endothelial antigen, survivin, tumor protein D52(TPD52), androgen receptor localization, ephrin type a receptor 2(EphA2), human telomerase reverse transcriptase (hTERT), survivin, mouse two minute 2 homolog (MDM2), cytochrome P4501B 1(CYP1B), HER2/neu, Wilms tumor gene 1(WT1), livin, Alpha Fetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16(MUC16), MUC1, Prostate Specific Membrane Antigen (PSMA), P53, or cyclin (D1) in the tumor vasculature.

In other aspects, the antigen can include a neoantigen. As used herein, the term "neoantigen" refers to an antigen encoded by a tumor-specific mutant gene.

In some aspects, the antigen is derived from a bacterium, a virus, a fungus, a protozoan, or any combination thereof. In some aspects, the antigen is derived from an oncogenic virus (also referred to herein as Cancer Associated Virus (CAV)). In other aspects, the antigen is derived from the group comprising: human gamma herpes virus 4 (i.e., Epstein Barr Virus (EBV)), influenza A virus, influenza B virus, cytomegalovirus, Staphylococcus aureus (staphylococcus aureus), Mycobacterium tuberculosis (mycobacterium tuberculosis), Chlamydia trachomatis (chlamydia trachomatis), HIV-1, HIV-2, coronaviruses (e.g., COVID-19, MERS-CoV, and SARS CoV), filoviruses (e.g., Marburg and Ebola), Streptococcus pyogenes (Streptococcus pyogenes), Streptococcus pneumoniae (Streptococcus pneumaoniae), Plasmodium species (e.g., Plasmodium vivax and Plasmodium falciparum), chikungunya virus, Human Papilloma Virus (HPV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), human T lymphocyte virus (LV 1), human HH virus (HHV8), and Klebsiella virus (MCV), Herpes simplex virus 2(HSV-2), certain species of Klebsiella sp, Pseudomonas aeruginosa, certain species of Enterococcus sp, certain species of Proteus sp, certain species of Enterobacter sp, certain species of Actinobacter sp, coagulase-negative staphylococci (CoNS), certain species of Mycoplasma sp, adenoviruses, adeno-associated viruses (AAV), or combinations thereof.

In some aspects, the EBV-derived antigen is BZLF 1. BZLF1 (also known as Zta or EB1) is an immediate early viral gene of EBV, can induce cancer and primarily infects B cells in 95% of the population. This gene (as well as other genes) leads to the expression of other EBV genes at other stages of disease progression and is involved in converting the virus from a latent form to a lytic form. ZEBRA (BamHI Z epstein-barr virus replication activators, also known as Zta and BZLF1) is an early lytic protein of EBV encoded by BZLF 1. See Hartlage et al (2015) Cancer Immunol. Res.3(7):787-94, and Rist et al (2015) J.virology 70:703-12, both of which are incorporated herein by reference in their entirety. EVs (e.g., exosomes) disclosed herein comprising an EBV antigen (e.g., BZLF1) may be used, for example, to treat post-transplant lymphoproliferative disorder (PTLD). Such EVs can be administered to EBV-negative patients receiving EBV-positive transplants. BZLF1 is a dominant T cell antigen and is associated with persistent remission in PTLD patients. EVs (e.g., exosomes) comprising BZLF1 disclosed herein can elicit effective CD8T cell-mediated immunity against BZLF 1. Thus, mucosal immunity and tissue resident memory cells can protect patients from developing PTLDF. Non-limiting exemplary antigens include, but are not limited to, the antigens disclosed in U.S. patent No. 8617564B 2.

In some aspects, the antigen is derived from mycobacterium tuberculosis to induce a cellular and/or humoral immune response. In some aspects, the antigen comprises one or more epitopes of mycobacterium tuberculosis (TB antigen). Several antigens are associated with mycobacterium tuberculosis infection, including ESAT-6, TB10.4, CFP10, Rv2031(hspX), Rv2654c (TB7.7) and Rv1038c (EsxJ). See, e.g., Lindestam et al, j.immunol.188(10):5020-31(2012), which is incorporated herein in its entirety. In some aspects, antigens useful in the present disclosure comprise one or more epitopes of ESAT 6. In some aspects, antigens useful in the present disclosure comprise one or more epitopes of TB 10.4. In some aspects, antigens useful in the present disclosure comprise one or more epitopes of CFP 10. In some aspects, antigens useful in the present disclosure comprise one or more epitopes of Rv2031 (hspX). In some aspects, antigens useful in the present disclosure comprise one or more epitopes of Rv2654c (TB 7.7). In some aspects, antigens useful in the present disclosure comprise one or more epitopes of Rv1038c (EsxJ). In some aspects, antigens useful in the present disclosure comprise an epitope selected from the group consisting of: ESAT6, TB10.4 (ESAT-6-like protein EsxH; CFP7), CFP10, Rv2031(hspX), Rv2654c (TB7.7), Rv1038c (EsxJ), and any combination thereof.

In some aspects, the TB antigen comprises a particular epitope of the TB antigen, e.g., a particular epitope of ESAT6 or TB 10.4. In some aspects, the ESAT6 antigen comprises an epitope of at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, at least ten amino acids, at least eleven amino acids, at least twelve amino acids, at least thirteen amino acids, at least fourteen amino acids, at least fifteen amino acids having the amino acid sequence set forth in MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA (SEQ ID NO: 230; GenBank: AWM 98862.1). In some aspects, wherein the TB10.4 antigen comprises an epitope of at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, at least ten amino acids, at least eleven amino acids, at least twelve amino acids, at least thirteen amino acids, at least fourteen amino acids, at least fifteen amino acids having the amino acid sequence set forth in the sequence seq id no: MSQIMYNYPAMLGHAGDMAGYAGTLQSLGAEIAVEQAALQSAWQGDTGITYQAWQAQWNQAMEDLVRAYHAMSSTHEANTMAMMARDPAEAAKWGG (SEQ ID NO: 231; NCBI reference sequence: WP _ 057308237.1).

In some aspects, the antigen comprises an autoantigen. As used herein, the term "autoantigen" refers to an antigen expressed by a host cell or tissue. Under normal health conditions, these antigens are recognized by the body as self and do not elicit an immune response. However, under certain disease conditions, the body's own immune system can recognize self-antigens as foreign and mount an immune response against them, resulting in autoimmunity. In certain aspects, EVs (e.g., exosomes) of the present disclosure may comprise autoantigens (i.e., self (germline) proteins for which a T cell response has been induced and which result in autoimmunity). Such EVs (e.g., exosomes) may be used to target autoreactive T cells and inhibit their activity. Non-limiting examples of autoantigens (including associated diseases or disorders) include: (i) beta-cell protein, insulin, islet antigen 2(IA-2), glutamate decarboxylase (GAD65) and zinc transporter 8(ZNT8) (type I diabetes mellitus), (ii) Myelin Oligodendrocyte Glycoprotein (MOG), Myelin Basic Protein (MBP), proteolipid protein (PLP) and Myelin Associated Glycoprotein (MAG) (multiple sclerosis), (iii) citrulline antigen and synovitin (rheumatoid arthritis), (IV) aquaporin-4 (AQP4) (neuromyelitis optica), (v) nicotinic acetylcholine receptors (nAChRs) (myasthenia gravis), (vi) desmoglein-1 (DSG1) and desmoglein-2 (DSG2) (pemphigus vulgaris), (v) thyroid stimulating hormone receptor (Graves disease), (vi) type IV collagen (Goodpasture's syndrome), (vii) thyroglobulin, thyroid peroxidase and Thyroid Stimulating Hormone Receptor (TSHR) (hashimoto's thyroiditis) or (viii) combinations thereof.

II.D.2 PROTAC

In some aspects, the payload comprises a proteolytic targeting chimera (PROTAC). PROTAC is a heterobifunctional molecule consisting of a ligand for a target protein, a ligand for E3 ubiquitinated ligase, and a linker connecting the two ligands. Once the target, the PROTAC E3 ternary complex is formed, E2 ubiquitin conjugating enzyme can transfer ubiquitin to lysine residues on the surface of the target protein. In some aspects, the PROTAC target is, e.g., era, BCR-ABL, BRD4, PDE4, ERR α, RIPK2, C-ABL, BRD2, BRD3, BRD4, FKBP12, TBK1, BRD9, EGFR, C-Met, Sirt2, CDK9, FLT3, BTK, ALK, AR, TRIM24, SMAD3, RAR, PI3K, PCAF, METAP2, HER2, HDAC6, GCN5, ERK1/2, DHODH, CRABP-II, FLT4, or CK 2. In some aspects, the PROTAC target ligand is, for example, 4-OHT, dasatinib, JQ1, a PDE4 inhibitor, JQ1, chloroalkane, a thiazolidinedione-based ligand, a RIPK2 inhibitor, bosutinib, a JQ1 derivative, OTX015, a steel factor, a TBK1 inhibitor, BI-7273, lapatinib, gefitinib, afatinib, fornitib, a Sirt2 inhibitor 3b, HJB97, SNS-032, an aminopyrazole analog, AC220, RN-486, ceritinib, an AR antagonist, IACS-7e, or an ibrutinib derivative. In some aspects, the PROTAC E3 ligand is, e.g., an LCL161 derivative, VHL1, hydroxyproline derivative, pomalidomide, thalidomide, HIF-1 alpha-derived (R) -hydroxyproline, VHL ligand 2, VH032 derivative, lenalidomide, thalidomide derivative, or VL-269. In some aspects, the E3 ligase is, for example, IAP, VHL, or CRBN. See, for example, An & Fu (2018) EBiomedicine 36:553-562, which is incorporated by reference herein in its entirety.

Procac and related technologies that can be used according to the methods disclosed herein are cited in patents such as WO2018106870, US2018155322, WO2018098288, WO2018098280, WO2018098275, WO2018089736, WO2018085247, US20180125821, US20180099940, WO2018064589, WO2018053354, WO 2017223423452, WO 201727201449, WO2017197056, WO2017197051, WO2017197046, WO 2012017103856, WO2017185034, WO2017185031, WO2017185023, WO 2017182828282418, US 20170767128170784, WO2017117474, WO2017117473, WO 20170723, US 993838383838264, US 20170065982016020170065982016033989, WO 2012012012012012012017072975632989, WO 2017272727272989, WO 2017272727298989, WO 20172727272727297989, WO 20172727272989, WO 2017272727297989, WO 2017297989, WO 201727272727272972049, WO 201727272972049, WO 2017272972049, WO 20172972043, WO 201727272727272727272727272972049, WO 20120072972049, WO 20120072989, WO 20172989, WO 20172727272727272727272727272972043, WO 20172727272727272972043, WO 2017272972043, WO 20172972043, WO 20172727272727272972043, WO 20172972043, WO 201727272727272727272972049, WO 201727272972043, WO 20120072972049, WO 2017272972049, WO 20172972043, WO 201200727272727272972049, WO 201200727272972043, WO 20120072972049, WO 20120072972043, WO 20172727272972043, WO 20172972043, WO 20120072972049, WO 201200727272727272972043, WO 20120072972049, WO 20120072972043, WO 201200727272972043, WO 20120072727272972043, WO 20120072972043, WO 20172972043, WO 20120072972043, WO 2017272727272972043, WO 20120072972043, WO 201727272727272972043, WO 20172972043, WO 2017272972043, WO 201727272727272727272727272972043, WO 20172972043, WO 20120072972043, WO 20072972043, WO 20120072972043, WO 201200729720429, WO 20120072972043, WO 201727272727272972043, WO 20120072727272972049, WO 20120072972043.

In some aspects, when several PROTACs are present on an EV (e.g., exosomes), such PROTACs may be the same or different. In some aspects, when several PROTACs are present on an EV (e.g., exosomes) disclosed herein, such PROTACs may be the same or different. In some aspects, an EV (e.g., exosome) composition of the present disclosure may comprise two or more EV (e.g., exosome) populations, wherein each EV (e.g., exosome) population comprises a different PROTAC or a combination thereof.

In some aspects, the PROTAC comprises at least one thiol group, wherein a maleimide moiety links the EV (e.g., exosome) to the PROTAC. In some aspects, the thiol group is located on the E3 ligase ligand portion of PROTAC. In some aspects, the thiol group is positioned on a target protein ligand portion of the ProTAC. In some aspects, the thiol group is positioned on the linker moiety of the PROTAC. In some aspects, the sulfhydryl group is a reactive group naturally present in PROTAC.

In other aspects, the maleimide moiety is introduced into the PROTAC, e.g., via chemical derivatization. In some aspects, chemical derivatization occurs via a bifunctional linker (bifunctional reagent) comprising a moiety capable of reacting with a chemical group present in the PROTAC, a moiety comprising a moiety capable of reacting with a maleimide moiety disclosed herein.

In some aspects, the E3 ligase ligand is linked to the PROTAC through a cleavable linker (e.g., PABC). In other aspects, the target ligand is linked to the PROTAC through a cleavable linker (e.g., PABC). In other aspects, both the E3 ligase ligand and the target ligand are linked to the PROTAC through a cleavable linker. In some aspects, the two cleavable linkers can be the same cleavable linker. In other aspects, the two cleavable linkers are different.

The function of a PROTAC (e.g., a PROTAC linked to an EV (e.g., an exosome disclosed herein)) can be assessed according to in vitro and in vivo methods known in the art. For example, as PROTAC induces ubiquitin-mediated degradation of a target protein, PROTAC activity can be determined using assays that directly measure target protein degradation (e.g., western blot) or measure target protein-mediated functional activity (e.g., phosphorylation or changes in phosphorylation-mediated cell signaling if the target protein is a protein kinase).

In some particular aspects, PROTAC comprises a TBK1 targeting ligand, a linker, and a VHL (E3 ligase) binding ligand (see, e.g., fig. 10C).

In other aspects, an EV (e.g., exosome) contains two precursors for forming CLIPTAC (click-formed PROTAC). Thus, an EV (e.g., an exosome of the present disclosure) may comprise two clitac precursor populations linked to the EV via a maleimide moiety. After uv light binds to the target cells, the cleptac precursors can be combined intracellularly by bioorthogonal click combining to produce heterobifunctional PROTACs. See Lebraud et al (2016) ACCCEnt. Sci.2: 927-one 934, which is incorporated herein by reference in its entirety.

In general, PROTAC can be described in terms of the formulas [ ULM ] - [ L ] - [ PTM ], where [ ULM ] is a ubiquitin-L binding moiety (first ligand), [ L ] is a linker, and [ PTM ] is a protein targeting moiety (second ligand). Exemplary PROTAC are shown in the table below. The table shows the ubiquitination enzymes targeted by [ ULM ] and its corresponding [ ULM ] ligand, and the proteins targeted by [ PTM ] and its corresponding [ ULM ] ligand.

Table 1: exemplary PROTAC

All other patent applications and patents disclosed in the above table are incorporated by reference in their entirety.

Specific linkers that may be used in the PROTAC are disclosed in, for example, U.S. patent application publication nos. US20180050021a1, US20180118733a1, US20180009779a1, US20180085465a1, US20180134684a1, and US20180134688a1, US patent nos. US9694084B2, US9821068B2, US9770512B2, and US9750816B2, and international application publication No. WO2018085247a1, the entire contents of which are incorporated herein by reference. WO2017212329a1 discloses the formation of PROTAC comprising a linker generated via a click reaction.

D.3 Interferon Gene stimulating protein (STING) agonists

In some aspects, the payload comprises nucleotides, wherein the nucleotides are interferon gene stimulating protein (STING) agonists. STING is a cytoplasmic sensor of cyclic dinucleotides, which are usually produced by bacteria. Upon activation, it leads to the production of type I interferons and elicits an immune response.

In some aspects, an EV (e.g., exosome) of the present disclosure comprises one or more STING agonists linked to the EV (e.g., exosome), e.g., chemically linked via a maleimide moiety. In some aspects, the STING agonist comprises a cyclic nucleotide STING agonist or an acyclic dinucleotide STING agonist.

Cyclic purine dinucleotides, such as, but not limited to, cGMP, cyclic di-GMP (c-di-GMP), cAMP, cyclic di-AMP (c-di-AMP), cyclic-di-GMP-AMP (cgamp), cyclic di-IMP (c-di-IMP), cyclic AMP-IMP (campi), and any analog thereof, are known to stimulate or enhance the immune or inflammatory response of a patient. The CDN may have a 2'2', 2'3', 2'5', 3'3' or 3'5' linkage connecting the cyclic dinucleotides, or any combination thereof.

Cyclic purine dinucleotides can be modified by standard organic chemistry techniques to produce analogues of purine dinucleotides. Suitable purine dinucleotides include, but are not limited to, adenine, guanine, inosine, hypoxanthine, xanthine, isoguanine, or any other suitable purine dinucleotide known in the art. The cyclic dinucleotide may be a modified analog. Any suitable modification known in the art may be used, including but not limited to phosphorothioate, phosphorodithioate, fluorinated, and difluorinated modifications.

Acyclic dinucleotide agonists, such as 5, 6-dimethylxanthone-4-acetic acid (DMXAA), or any other acyclic dinucleotide agonist known in the art may also be used.

It is contemplated that any STING agonist may be used. Among the STING agonists are DMXAA, STING agonist-1, ML RR-S2CDA, ML RR-S2c-di-GMP, ML-RR-S2cGAMP, 2'3' -c-di-AM (PS)2, 2'3' -cGAMP, 2'3' -cGAMPdFHS, 3'3' -cGAMP, 3'3' -cGAMPFSH, cAIMP, cAIM (PS)2, 3'3' -cAIMP, 3'3' -cAIMPdFSH, 2 '2' -cGAMP, 2'3' -cGAM (PS)2, 3'3' -cGAMP, c-di-AMP, 2'3' -c-di-AM (PS)2, c-di-GMP, 2'3' -c-di-GMP, c-di-IMP, c-di-UMP or any combination thereof. In a particular aspect, the STING agonist is 3'3' -caimdfsh, alternatively designated 3-3 caimdfsh. Other STING agonists known in the art may also be used.

In some aspects, STING agonists useful in the present disclosure include compounds selected from the group consisting of:

in some aspects, STING agonists useful in the present disclosure include compounds having the formula:

wherein:

X₁h, OH or F;

X₂h, OH or F;

z is OH, OR₁SH or SR₁Wherein:

R₁Is Na or NH₄Or is or

R₁Is an enzyme labile group that provides OH or SH in vivo, such as pivaloyloxymethyl;

bi and B2 are bases selected from:

the precondition is that:

-in formula 1: x₁And X₂Is not an OH group, but is a group,

-in formula 2: when X is present₁And X₂When is OH, B₁Is not adenine and B₂Is not guanine, and

-in formula 3: when X is present₁And X₂When it is OH, B₁Not adenine, B₂Is not guanine and Z is not OH. See WO 2016/096174, the contents of which are incorporated herein by reference in their entirety.

In some aspects, STING agonists useful in the present disclosure include:

a pharmaceutically acceptable salt thereof. See WO 2016/096174a 1.

In other aspects, STING agonists useful in the present disclosure include compounds having the formula:

or any pharmaceutically acceptable salt thereof.

In some aspects, STING agonists useful in the present disclosure include compounds having the formula:

wherein each symbol is defined in WO 2014/093936 (the contents of which are incorporated herein by reference in their entirety).

In some aspects, STING agonists useful in the present disclosure include compounds having the formula:

wherein each substituent is defined in WO 2014/189805 (the contents of which are incorporated herein by reference in their entirety).

In some aspects, STING agonists useful in the present disclosure include compounds having the formula:

wherein each substituent is defined in WO 2015/077354 (the contents of which are incorporated herein by reference in their entirety). See also Cell ports 11,1018-1030 (2015).

In some aspects, STING agonists useful in the present disclosure include c-di-AMP, c-di-GMP, c-di-IMP, c-AMP-GMP, c-AMP-IMP, and c-GMP-IMP, as described in WO2013/185052 and sci. trans. med.283,283ra52(2015), which are incorporated herein by reference in their entirety.

In some aspects, STING agonists useful in the present disclosure include compounds having a formula selected from

Wherein each substituent (i.e., R1, R2, R3, R4, and X) is defined in WO 2014/189806 (the contents of which are incorporated herein by reference in their entirety).

In some aspects, STING agonists useful in the present disclosure include compounds having the formula:

wherein each substituent (i.e., R1, R2, R3, R4, R5, R6, Y1, and Y2) is defined in WO 2015/185565 (the contents of which are incorporated herein by reference in their entirety).

In some aspects, STING agonists useful in the present disclosure include compounds selected from the following formulae:

wherein each substituent (i.e., X and Y) is defined in WO 2014/179760 (the contents of which are incorporated herein by reference in their entirety).

In some aspects, STING agonists useful in the present disclosure include compounds having one of the following formulae:

wherein each substituent (i.e., R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, Xa1, Xb1, Xc, Xd, Xe and Xf) is defined in WO 2014/179335 (the contents of which are incorporated herein by reference in their entirety).

In some aspects, STING agonists useful in the present disclosure include compounds having the formula:

it is described in WO 2015/017652 (the contents of which are incorporated herein by reference in their entirety).

In some aspects, STING agonists useful in the present disclosure include compounds having the formula:

it is described in WO 2016/096577 (the contents of which are incorporated herein by reference in their entirety).

In some aspects, STING agonists useful in the present disclosure include compounds having the formula:

wherein each substituent (i.e., R1, R2, R3, R4, R5 and R6) is described in WO2011/003025 (the contents of which are incorporated herein by reference in their entirety).

In some aspects, STING agonists useful in the present disclosure include compounds having the formula:

wherein each substituent (i.e., R1, R2, R3, R4, R5 and R6) is defined in WO2016/145102 (the contents of which are incorporated herein by reference in their entirety).

In some aspects, STING agonists useful in the present disclosure include compounds having the formula:

wherein each substituent is defined in WO 2017/027646 (the contents of which are incorporated herein by reference in their entirety).

In some aspects, STING agonists useful in the present disclosure include compounds having the formula:

wherein each substituent is defined in WO 2017/075477 (the contents of which are incorporated herein by reference in their entirety).

In some aspects, STING agonists useful in the present disclosure include compounds having the formula:

wherein each substituent is defined in WO 2017/027645 (the contents of which are incorporated herein by reference in their entirety).

In some aspects, STING agonists useful in the present disclosure include compounds having the formula:

wherein each substituent is defined in WO 2018/100558 (the contents of which are incorporated herein by reference in their entirety).

In some aspects, STING agonists useful in the present disclosure include compounds having the formula:

wherein each substituent is defined in WO 2017/175147 (the contents of which are incorporated herein by reference in their entirety).

In some aspects, STING agonists useful in the present disclosure include compounds having the formula:

Wherein each substituent is defined in WO 2017/175156 (the contents of which are incorporated herein by reference in their entirety).

In some aspects, the EV (e.g., exosomes) comprise a cyclic-dinucleotide STING agonist and/or an acyclic-dinucleotide STING agonist. In some aspects, when several cyclic-dinucleotide STING agonists are present on an EV (e.g., exosomes) disclosed herein, such STING agonists may be the same, or they may be different. In some aspects, when several non-cyclic dinucleotide STING agonists are present, such STING agonists may be the same, or they may be different. In some aspects, an EV (e.g., exosome) composition of the present disclosure may comprise two or more EV (e.g., exosome) populations, wherein each EV (e.g., exosome) population comprises a different STING agonist or a combination thereof.

In some particular aspects, an EV (e.g., exosome) of the present disclosure comprises (MM) - (linker) - (bioactive molecule) having formula (IV):

in some particular aspects, an EV (e.g., exosome) of the present disclosure comprises (MM) - (linker) - (bioactive molecule) having formula (V):

in some particular aspects, the EVs (e.g., exosomes) of the present disclosure comprise a compound (CP249) having formula (VI):

In some particular aspects, the EVs (e.g., exosomes) of the present disclosure comprise a compound (CP250) having formula (VII):

in some particular aspects, an EV (e.g., exosome) of the present disclosure comprises a compound (CP260) having formula (VIII):

in some particular aspects, an EV (e.g., exosome) of the present disclosure comprises a compound (CP261) having formula (IX):

in some aspects, STING agonists that may be used in the EV conjugates of the invention include, but are not limited to, CP247, CP250, CP260, CP261, or pharmaceutically acceptable salts thereof. In some aspects, STING agonists useful in the EV conjugates of the invention include CP247 or a pharmaceutically acceptable salt thereof. In some aspects, STING agonists useful in the EV conjugates of the invention include CP250, or a pharmaceutically acceptable salt thereof. In some aspects, STING agonists useful in the EV conjugates of the invention include CP260 or a pharmaceutically acceptable salt thereof. In some aspects, STING agonists useful in the EV conjugates of the invention include CP261 or a pharmaceutically acceptable salt thereof.

In other aspects, STING agonists that can be used in the EV conjugates of the invention include, but are not limited to, CP227, CP229, or pharmaceutically acceptable salts thereof. In other aspects, STING agonists useful in the EV conjugates of the invention include CP227 or a pharmaceutically acceptable salt thereof. In other aspects, STING agonists that can be used in the EV conjugates of the invention include, but are not limited to, CP229 or a pharmaceutically acceptable salt thereof.

D.4 TLR agonists

In some aspects, the payload comprises a TLR agonist. Non-limiting examples of TLR agonists include: TLR2 agonists (e.g., lipoteichoic acid, atypical LPS, MALP-2 and MALP-404, OspA, porin, LcrV, lipidated mannan, GPI, lysophosphatidylserine, Lipophoglycan (LPG), Glycophosphatidylinositol (GPI), zymosan, hsp60, gH/gL glycoprotein, hemagglutinin), TLR3 agonists (e.g., double-stranded RNA, e.g., poly (I: C)), TLR4 agonists (e.g., Lipopolysaccharide (LPS), lipoteichoic acid, β -defensin 2, fibronectin EDA, HMGB1, snapin, tenascin C), TLR5 agonists (e.g., flagellin), TLR6 agonists, TLR7/8 agonists (e.g., single-stranded RNA, a, poly G10, poly G3, ranisimo CpG), TLR9 agonists (e.g., unmethylated DNA), and combinations thereof. Non-limiting examples of TLR agonists can be found in WO2008115319a2, US20130202707a1, US20120219615a1, US20100029585a1, WO2009030996a1, WO2009088401a2, and WO2011044246a1, each of which is incorporated by reference in its entirety.

II.D.5 antibodies

In some aspects, the payload comprises an antibody or antigen-binding fragment thereof. In some aspects, the payload includes an ADC. In some aspects, the payload comprises a small molecule comprising a synthetic antineoplastic agent (e.g., monomethyl auristatin e (mmae) (vedotin)), an inhibitor of cytokine release (e.g., MCC950), an mTOR inhibitor (e.g., rapamycin and its analogs (Rapalog), an autotaxin inhibitor (e.g., PAT409), a lysophosphatidic acid receptor antagonist (e.g., AM152, also known as BMS-986020), or any combination thereof.

Bioactive molecules targeting macrophages

In some aspects, the payload comprises a bioactive molecule that targets macrophages. In other aspects, the payload comprises a biologically active molecule that induces macrophage polarization. Macrophage polarization is the process by which macrophages adopt different functional programs in response to signals from the macrophage microenvironment. This ability is associated with multiple roles of macrophages in the organism: macrophages are potent effector cells of the innate immune system and are also important in cell debris removal, embryo development, and tissue repair.

By simplifying the classification, macrophage phenotypes have been divided into 2 groups: m1 (classical activated macrophages) and M2 (alternative activated macrophages). This rough classification is based on in vitro studies in which cultured macrophages are treated with molecules that stimulate their phenotypic switch to a specific state. In addition to chemical stimulation, the stiffness of the underlying matrix for macrophage growth has been shown to direct polarization state, functional effects, and migration patterns. M1 macrophages are described as a pro-inflammatory type and play an important role in host defense directly against pathogens, such as phagocytosis and secretion of pro-inflammatory cytokines and microbicidal molecules. M2 macrophages are described as having the exact opposite function: regulating the resolution phase of inflammation and repairing damaged tissues. Later, more extensive in vitro and ex vivo studies showed that macrophage phenotypes were more diverse, overlapping each other in gene expression and function, revealing that these many mixed states form a continuous activation state that is dependent on the microenvironment. Furthermore, in vivo, the gene expression profiles between different tissue macrophage populations are highly diverse. Therefore, the macrophage activation profile is considered to be more extensive, involving complex regulatory pathways that respond to a plethora of diverse signals from the environment. The diversity of macrophage phenotypes remains to be well characterized in vivo.

An imbalance in macrophage types is associated with many immune-related diseases. For example, an increased ratio of M1/M2 may be associated with the development of inflammatory bowel disease and obesity in mice. In another aspect, in vitro experiments indicate that M2 macrophages are the primary mediator of tissue fibrosis. Several studies have linked the fibrotic characteristics of M2 macrophages to the pathogenesis of systemic sclerosis. Non-limiting examples of macrophages targeting biologically active molecules are: PI3K γ (phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit γ), RIP1 (receptor interacting protein (RIP) kinase 1, RIPK1), HIF-1 α (hypoxia inducible factor 1- α), AHR1 (adhesion and hyphal regulatory factor 1), miR146a, miR155, IRF4 (interferon regulatory factor 4), PPAR γ (peroxisome proliferator activated receptor γ), IL-4RA (interleukin 4 receptor subunit α), TLR8 (Toll-like receptor 8), and TGF- β 1 (transforming growth factor β -1 preprotein).

In some aspects, the payload comprises a biologically active molecule that targets a PI3K gamma protein or transcript (PI3K gamma antagonist). In some aspects, the PI3K γ antagonist is an antisense oligonucleotide. In other aspects, the PI3K γ antagonist is a small molecule. In some aspects, the ASO targets a transcript encoding PI3K γ, e.g., mRNA. The sequence of the PI3K γ gene can be found at chromosome position 7q22.3 and publicly available GenBank accession No. NC _000007.14(106865282..106908980), which is incorporated by reference in its entirety. The sequence of the human PI3K γ protein can be found in publicly available UniProt accession number P48736, which is incorporated by reference herein in its entirety.

In some aspects, the payload comprises a biologically active molecule that targets the RIP1 protein or transcript (RIP1 antagonist). In some aspects, the RIP1 antagonist is an antisense oligonucleotide. In other aspects, the RIP1 antagonist is a small molecule. In some aspects, the ASO targets a transcript, e.g., mRNA, encoding RIP 1. The sequence of the RIP1 gene can be found at chromosome position 6p25.2 and publicly available GenBank accession No. NC _000006.12(3063967..3115187), which is incorporated by reference in its entirety. The sequence of human RIP1 protein can be found in publicly available UniProt accession No. Q13546, which is incorporated by reference herein in its entirety.

In some aspects, the payload comprises a biologically active molecule that targets a HIF-1 α protein or transcript (HIF-1 α antagonist). In some aspects, the HIF-1 α antagonist is an antisense oligonucleotide. In other aspects, the HIF-1 α antagonist is a small molecule. In some aspects, an ASO targets a transcript, e.g., mRNA, encoding HIF-1 α. The sequence of the HIF-1 α gene can be found at chromosome position 14q23.2 and publicly available GenBank accession No. NC _000014.9(61695513..61748259), which is incorporated by reference in its entirety. The sequence of the human HIF-1. alpha. protein can be found in publicly available UniProt accession No. Q16665, which is incorporated by reference herein in its entirety. In some aspects, the ASO targets an mRNA encoding HIF-2 α. The sequence of the HIF-2 α gene can be found at chromosomal position 2p21 and publicly available GenBank accession No. NC _000002.12(46297407..46386697), which is incorporated by reference in its entirety. The sequence of the human HIF-2. alpha. protein can be found in publicly available UniProt accession number Q99814, which is incorporated by reference herein in its entirety.

In some aspects, the payload comprises a biologically active molecule that targets an AHR1 protein or transcript (AHR1 antagonist). In other aspects, the AHR1 antagonist is a small molecule.

In some aspects, the payload comprises a biologically active molecule targeting miR146a (miR146a antanemia). In some aspects, miR146a is an antisense oligonucleotide. In some aspects, the ASO binds to miR146a-5p (ugagaacugaucauggguu) (SEQ ID NO: 226). In some aspects, the ASO binds to miR146a-3p (cccuguaauucaguucag) (SEQ ID NO: 227).

In some aspects, the payload comprises a biologically active molecule that mimics miR155 (a miR155 mimetic). In some aspects, the miR155 mimetic is RNA or DNA. In some aspects, the miR155 mimetic comprises the nucleotide sequence of miR155-5p (uuaaugcuaaucgugauaggggu) (SEQ ID NO: 228). In some aspects, the miR155 mimetic comprises the nucleotide sequence of miR155-3p (cuccuacauauuagcauuaaca) (SEQ ID NO: 229).

In some aspects, the payload comprises a biologically active molecule that targets an IRF-4 protein or transcript (IRF4 antagonist). In some aspects, the IRF4 antagonist is an antisense oligonucleotide. In some aspects, the ASO targets a transcript encoding IRF-4, e.g., mRNA. The sequence of the IRF-4 gene can be found at chromosome position 6p25.3 and publicly available GenBank accession No. NC _000006.12(391739..411443), which is incorporated by reference in its entirety. The sequence of the human IRF-4 protein can be found in publicly available UniProt accession number Q15306, which is incorporated by reference herein in its entirety.

In some aspects, the payload comprises a biologically active molecule that targets a PPAR γ protein or transcript (PPAR γ antagonist). In some aspects, the PPAR γ antagonist is an antisense oligonucleotide. In other aspects, the PPAR γ antagonist is a small molecule. In some aspects, the ASO targets a transcript encoding PPAR γ, e.g., mRNA. The sequence of the PPAR γ gene can be found in chromosome position 3p25.2 and publicly available GenBank accession No. NC _000003.12(12287485..12434356), which is incorporated by reference in its entirety. The sequence of the human PPAR γ protein can be found in publicly available UniProt accession number P37231, which is incorporated by reference herein in its entirety.

In some aspects, the payload comprises a bioactive molecule that targets an IL-4RA protein or transcript (IL-4RA antagonist). In some aspects, the IL-4RA antagonist is an antisense oligonucleotide. In some aspects, the ASO targets a transcript encoding IL-4RA, e.g., mRNA. The sequence of the IL-4RA gene can be found at chromosome position 16p12.1 and publicly available GenBank accession No. NC _000016.10(27313668..27364778), which is incorporated by reference in its entirety. The sequence of the human IL-4RA protein can be found in publicly available UniProt accession number P24394, which is incorporated by reference herein in its entirety.

In some aspects, the payload comprises a biologically active molecule that is an agonist of Toll-like receptor 8(TLR 8). TLR8 is also known as CD 288. TLR8 is a key component of innate and adaptive immunity. TLRs (Toll-like receptors) control the host immune response to pathogens by recognizing molecular patterns unique to microorganisms. It acts through MYD88 and TRAF6, resulting in NF- κ -B activation, cytokine secretion and inflammatory responses. The sequence of the human TLR8 protein can be found in publicly available UniProt accession No. Q9NR97, which is incorporated by reference herein in its entirety.

In some aspects, the payload comprises a biologically active molecule that targets a TGF- β 1 protein or transcript (a TGF- β 1 antagonist). In some aspects, the TGF- β 1 antagonist is an antisense oligonucleotide. In some aspects, the ASO targets a transcript encoding TGF- β 1, e.g., mRNA. The sequence of the TGF- β 1 gene can be found in chromosomal location 19q13.2 and publicly available GenBank accession No. NC _000019.10(41330323..41353922, complement), which is incorporated by reference in its entirety. The sequence of the human TGF- β 1 protein can be found in the publicly available UniProt accession number P01137, which is incorporated by reference in its entirety. ASOs may comprise one or more nucleosides having a modified sugar moiety, i.e., a modification of the sugar moiety when compared to the ribose moiety present in DNA and RNA. Nucleosides have been prepared with many modifications of the ribose moiety, the primary purpose being to improve certain properties of the oligonucleotide, such as affinity and/or nuclease resistance. Such modifications include those in which the ribose ring structure is modified, for example by substitution with a hexose ring (HNA) or a bicyclic ring, which typically has a diradical bridge between the C2 'and C4' carbons on the ribose ring (LNA), or an unconnected ribose ring, which typically lacks a bond between the C2 'and C3' carbons (e.g., UNA). Other sugar-modified nucleosides include, for example, bicyclic hexose nucleic acids (WO2011/017521) or tricyclic nucleic acids (WO 2013/154798). Modified nucleosides also include nucleosides in which the sugar moiety is replaced with a non-sugar moiety, for example in the case of Peptide Nucleic Acid (PNA) or morpholino nucleic acid. Sugar modifications also include modifications by altering substituents on the ribose ring to groups other than hydrogen or to the naturally occurring 2' -OH groups in RNA nucleosides. For example, substituents may be introduced at the 2', 3', 4 'or 5' positions. Nucleosides having modified sugar moieties also include 2 'modified nucleosides, such as 2' substituted nucleosides. In fact, much attention has been focused on the development of 2 'substituted nucleosides, and many 2' substituted nucleosides were found to have beneficial properties when incorporated into oligonucleotides, such as enhanced nucleoside resistance and enhanced affinity. 2' sugar modified nucleosides are nucleosides having a substituent other than H or-OH at the 2' position (2' substituted nucleosides) or containing a 2' linked diradical, and include 2' substituted nucleosides and LNA (2' -4' diradical bridged) nucleosides. For example, 2 'modified sugars can provide enhanced binding affinity (e.g., enhanced affinity 2' sugar modified nucleosides) and/or enhanced nuclease resistance to oligonucleotides. Examples of 2' -substituted modified nucleosides are 2' -O-alkyl-RNA, 2' -O-methyl-RNA, 2' -alkoxy-RNA, 2' -O-methoxyethyl-RNA (MOE), 2' -amino-DNA, 2' -fluoro-RNA, 2' -fluoro-DNA, arabinonucleic acid (ANA), and 2' -fluoro-ANA nucleosides. For additional examples, see, e.g., Freier & Altmann; nucleic acid Res.,1997,25, 4429-4443; uhlmann, curr. opinion in Drug Development,2000,3(2), 293-; and Deleavey and Damha, Chemistry and Biology 2012,19, 937. Below are schematic representations of some 2' substituted modified nucleosides.

LNA nucleosides are modified nucleosides that contain a linker group (called a diradical or bridge) between C2 'and C4' of the ribose ring (i.e., the 2 '-4' bridge) of the nucleoside that restricts or locks the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged or Bicyclic Nucleic Acids (BNA). The locking of the ribose conformation is associated with enhanced hybridization affinity (duplex stability) when LNA is incorporated into an oligonucleotide of a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide/complementary duplex.

Non-limiting exemplary LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352, WO 2004/046160, WO 00/047599, WO 2007/134181, WO 2010/077578, WO 2010/036698, WO 2007/090071, WO 2009/006478, WO 2011/156202, WO 2008/154401, WO 2009/067647, WO 2008/150729, Morita et al, Bioorganic & Med.chem.Lett.12,73-76, Seth et al, J.org.chem.2010, Vol.75 (5), pp.1569-81 and Mitsuoka et al, Nucleic Acids Research 2009,37(4), 1225-1238.

In some aspects, the modified nucleoside or LNA nucleoside of the ASO of the present disclosure has the general structure of formula I or II:

wherein

W is selected from-O-, -S-, -N (R) ^a)-、-C(R^aR^b) -, in particular-O-;

b is a nucleobase or a modified nucleobase moiety;

z is an internucleoside linkage to an adjacent nucleoside or 5' -terminal group;

z is an internucleoside linkage to an adjacent nucleoside or 3' -terminal group;

R¹、R²、R³、R⁵and R^5*Independently selected from the group consisting of hydrogen, halogen, alkyl, alkenyl, alkynyl, hydroxy, alkoxy, alkoxyalkyl, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl, formyl, azide, heterocycle, and aryl; and is

X、Y、R^aAnd R^bAs defined herein.

II.D.7 oligonucleotides

In some aspects, the payload comprises an antisense oligonucleotide, diamidophosphomorpholino oligomer (PMO), or peptide-conjugated diamidomorpholino oligomer (PPMO), an antisense oligonucleotide (ASO), an siRNA, an miRNA, an shRNA, a nucleic acid, or any combination thereof.

In some aspects, the ASO is a PI3K γ antagonist. In some aspects, the ASO targets a transcript encoding PI3K γ, e.g., mRNA. The sequence of the PI3K γ gene can be found at chromosome position 7q22.3 and publicly available GenBank accession No. NC _000007.14(106865282..106908980), which is incorporated by reference in its entirety. The sequence of the human PI3K γ protein can be found in publicly available UniProt accession number P48736, which is incorporated by reference herein in its entirety.

In some aspects, the ASO is a RIP1 antagonist. In some aspects, the ASO targets a transcript, e.g., mRNA, encoding RIP 1. The sequence of the RIP1 gene can be found at chromosome position 6p25.2 and publicly available GenBank accession No. NC _000006.12(3063967..3115187), which is incorporated by reference in its entirety. The sequence of human RIP1 protein can be found in publicly available UniProt accession No. Q13546, which is incorporated by reference herein in its entirety.

In some aspects, the ASO is an HIF-1 α antagonist. In some aspects, an ASO targets a transcript, e.g., mRNA, encoding HIF-1 α. The sequence of the HIF-1 α gene can be found at chromosome position 14q23.2 and publicly available GenBank accession No. NC _000014.9(61695513..61748259), which is incorporated by reference in its entirety. The sequence of the human HIF-1. alpha. protein can be found in publicly available UniProt accession No. Q16665, which is incorporated by reference herein in its entirety. In some aspects, the ASO targets an mRNA encoding HIF-2 α. The sequence of the HIF-2 α gene can be found at chromosomal position 2p21 and publicly available GenBank accession No. NC _000002.12(46297407..46386697), which is incorporated by reference in its entirety. The sequence of the human HIF-2. alpha. protein can be found in publicly available UniProt accession number Q99814, which is incorporated by reference herein in its entirety.

In some aspects, the ASO is an IRF4 antagonist. In some aspects, the ASO targets a transcript encoding IRF-4, e.g., mRNA. The sequence of the IRF-4 gene can be found at chromosome position 6p25.3 and publicly available GenBank accession No. NC _000006.12(391739..411443), which is incorporated by reference in its entirety. The sequence of the human IRF-4 protein can be found in publicly available UniProt accession number Q15306, which is incorporated by reference herein in its entirety.

In some aspects, the ASO is a PPAR γ antagonist. In some aspects, the ASO targets a transcript encoding PPAR γ, e.g., mRNA. The sequence of the PPAR γ gene can be found in chromosome position 3p25.2 and publicly available GenBank accession No. NC _000003.12(12287485..12434356), which is incorporated by reference in its entirety. The sequence of the human PPAR γ protein can be found in publicly available UniProt accession number P37231, which is incorporated by reference herein in its entirety.

In some aspects, the ASO is an IL-4RA antagonist. In some aspects, the ASO targets a transcript encoding IL-4RA, e.g., mRNA. The sequence of the IL-4RA gene can be found at chromosome position 16p12.1 and publicly available GenBank accession No. NC _000016.10(27313668..27364778), which is incorporated by reference in its entirety. The sequence of the human IL-4RA protein can be found in publicly available UniProt accession number P24394, which is incorporated by reference herein in its entirety.

In some aspects, the ASO is a TGF- β 1 antagonist. In some aspects, the ASO targets a transcript encoding TGF- β 1, e.g., mRNA. The sequence of the TGF- β 1 gene can be found in chromosomal location 19q13.2 and publicly available GenBank accession No. NC _000019.10(41330323..41353922, complement), which is incorporated by reference in its entirety. The sequence of the human TGF- β 1 protein can be found in the publicly available UniProt accession number P01137, which is incorporated by reference in its entirety.

In some aspects, the ASO targets a transcript that is a STAT6 transcript, a CEBP/β transcript, a STAT3 transcript, a KRAS transcript, an NRAS transcript, an NLPR3 transcript, or any combination thereof.

STAT6(STAT6) is also known as a signal transducer and transcriptional activator 6. Synonyms for STAT6/STAT6 are known, including IL-4 STATs; STAT, interleukin 4 induction; the transcription factor IL-4 STAT; STAT 6B; STAT 6C; and D12S 1644. The sequence of the human STAT6 gene can be found in publicly available GenBank accession numbers NC-000012.12: c 57111413-57095404. The human STAT6 gene can be found in 57111413-57095404, the complement of chromosomal location 12q 13.3.

CEBP/β (CEBP/β) is also known as CCAAT/enhancer binding protein β. Synonyms for CEBP/β/CEBP/β are known, including C/EBP β; a liver activating protein; LAP; suppressor proteins enriched in liver; LIP; nuclear factor NF-IL 6; a transcription factor 5; TCF-5; CEBP; CEBPb; CEBP β; CEBP/B; and TCF 5. The sequence of the human CEBP/β gene can be found in publicly available GenBank accession No. NC _000020.11(50190583.. 50192690). The human CEBP/β gene can be found in 50190583-50192690 of chromosomal location 20q 13.13.

NRas is an oncogene that encodes a membrane protein that shuttles between the golgi apparatus and the plasma membrane. Genomic DNA encoding NRas can be found at chromosomal position 1p13.2 (i.e., nucleotides 5001 to 17438 of GenBank accession No. NG _ 007572). N-ras mutations have been described in melanoma, thyroid cancer, teratocarcinoma, fibrosarcoma, neuroblastoma, rhabdomyosarcoma, burkitt lymphoma, acute promyelocytic leukemia, T-cell leukemia, and chronic myelocytic leukemia. Oncogenic N-Ras induces Acute Myeloid Leukemia (AML) or chronic myelomonocytic leukemia (CMML) -like disease in mice. The neuroblastoma RAS virus oncogene (NRas) has a number of names in the art. Such names include: GTPase NRas, part 4 of the N-RAS protein, a neuroblastoma RAS virus oncogene homolog, a transforming protein N-RAS and a v-RAS neuroblastoma RAS virus oncogene homolog.

Signal transducer and activator of transcription 3(STAT3) is a signal transducer and activator of transcription that can transport signals from cell surface receptors to the nucleus of the cell. STAT3 is often over-activated in many human cancers. Genomic DNA encoding STAT3 can be found at chromosome position 17q21.2 (i.e., nucleotides 5,001 to 80,171 of GenBank accession No. NG _ 007370.1).

NLRP3(NLRP3) is also known as NLR family pyrin-containing domain 3. Synonyms for NLRP3/NLRP3 are known, including NLRP 3; c1orf 7; CIAS 1; NALP 3; PYPAF 1; a nucleotide-binding oligomerization domain, a leucine-rich repeat, and a pyrin-containing domain 3; cold-induced autoinflammatory syndrome 1 protein; freezing the protein; protein 3 containing NACHT, LRR and PYD domains; angiotensin/vasopressin receptor AII/AVP-like; 1.1 of caterpillar protein; CLR 1.1; cold-induced autoinflammatory syndrome 1 protein; and PYRIN-containing APAF 1-like protein 1. The sequence of the human NLRP3 gene can be found in publicly available GenBank accession No. NC-000001.11: 247416156-. The human NLRP3 gene can be found in 247,416,156-247,449,108 of chromosome position 1q 44.

KRAS has several names in the art. These names include: KRAS protooncogene, GTPase; V-Ki-Ras2Kirsten rat sarcoma 2 viral oncogene homolog; (iii) a GTP enzyme KRas; C-Ki-Ras; K-Ras 2; KRAS 2; RASK 2; V-Ki-Ras2Kirsten rat sarcoma virus oncogene homolog; kirsten rat sarcoma virus protooncogene; transforming a protooncogene in a cell; the cellular C-Ki-Ras2 protooncogene; a transforming protein P21; PR310C-K-Ras oncogene; C-Kirsten-Ras protein; K-Ras P21 protein; and the oncogene KRAS 2. The sequence of the human KRAS gene can be found at chromosomal location 12p12.1 and publicly available GenBank accession number NC-000012 (25,204, 789-. The genomic sequence of the human wild-type KRAS transcript corresponds to the reverse complement of residues 25,204, 789-25, 250,936 of NC-000012

II.D.8 Transporter peptides

In some aspects, the payload comprises a cell transit peptide, a cell penetrating peptide, or a fusion peptide. As used herein, the term "transit peptide" refers to any peptide sequence that facilitates the movement of any attached cargo within one or more cells, including peptide sequences that facilitate the passage of cargo across the cell membrane of a cell, the secretion of cargo from a cell or EV, and the release of cargo from a cell or EV, as well as other modes of cell movement. In specific but non-limiting examples, the transit peptide may be a sequence derived from a cell penetrating peptide, a non-canonical secretory sequence, an endosomal release domain, a receptor binding domain, and a fusion peptide.

As used herein, the term "cell penetrating peptide" refers to any peptide sequence that facilitates the movement of any attached cargo across a lipid bilayer, such as a cell membrane or EV membrane. As used herein, the term "non-canonical secretory sequence" refers to any protein or peptide sequence that provides for secretion of any attached cargo from a cell by the ER-golgi independent pathway. As used herein, the term "endosomal release domain" refers to any peptide sequence that facilitates the release of any attached cargo from the endosome of a cell or EV. As used herein, the term "receptor binding domain" refers to any RNA or protein domain that is capable of interacting with a surface-bound cellular receptor. As used herein, the term "fusion peptide" refers to any peptide sequence that facilitates the export of cargo from an EV or cell.

In some aspects, an EV (e.g., exosome) of the present disclosure comprises a transit peptide and a second payload, e.g., another biologically active molecule, such as a polynucleotide (e.g., antisense oligonucleotide or interfering RNA).

II.D.9 adeno-associated virus (AAV)

In some aspects, the payload comprises an adeno-associated virus (AAV). In some aspects, the AAV is linked (e.g., chemically linked via a maleimide moiety) to a cavity surface of the EV. In some aspects, the AAV is linked (e.g., chemically linked via a maleimide moiety) to the exterior surface of the EV. In some aspects, the AAV is linked (e.g., chemically linked via a maleimide moiety) to a scaffold, e.g., a protein scaffold, such as a protein X scaffold or fragment thereof, or to a lipid scaffold (e.g., cholesterol). In some aspects, the AAV is chemically linked to the scaffold moiety via a reaction between a maleimide group present on the AAV capsid protein and a thiol group present on the scaffold (e.g., naturally occurring or introduced through a linker or bifunctional reagent). In some aspects, the AAV is chemically linked to the scaffold moiety via a reaction between a thiol group present on the AAV capsid protein (e.g., naturally occurring or introduced through a linker or bifunctional reagent) and a maleimide group present on the scaffold moiety.

In some aspects, the AAV comprises a genetic cassette. In some aspects, the genetic cassette encodes a protein selected from the group consisting of: secreted proteins, receptors, structural proteins, signaling proteins, sensory proteins, regulatory proteins, transport proteins, storage proteins, defense proteins, motor proteins, clotting factors, growth factors, antioxidants, cytokines, chemokines, enzymes, tumor suppressor genes, DNA repair proteins, structural proteins, low density lipoprotein receptors, alpha glucosidase, cystic fibrosis transmembrane conductance regulator, or any combination thereof. In some aspects, the genetic cassette encodes a factor VIII protein or a factor IX protein. In some aspects, the factor VIII protein is wild-type factor VIII, B-domain deleted factor VIII, a factor VIII fusion protein, or any combination thereof.

In some aspects, the AAV is selected from the group consisting of: AAV type 1, AAV type 2, AAV type 3A, AAV type 3B, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, synthetic AAV, any combination thereof.

II.D.10 immunomodulators

In some aspects, the payload comprises an immunomodulatory agent. In certain aspects, an immunomodulatory agent is linked (e.g., chemically linked via a maleimide moiety) to a scaffold moiety, e.g., a scaffold X protein or fragment thereof, on the outer surface of an EV (e.g., exosome) or on the luminal surface of an EV (e.g., exosome). In some aspects, an immunomodulatory agent is linked (e.g., chemically linked via a maleimide moiety) to a scaffold moiety, such as a scaffold Y protein or fragment thereof, on the luminal surface of an EV (e.g., an exosome). In other aspects, the immunomodulator is in the lumen of an EV (e.g., exosome).

In some aspects, the immune modulator comprises an inhibitor of a negative checkpoint modulator or an inhibitor of a binding partner of a negative checkpoint modulator. In certain aspects, the negative checkpoint modulator comprises: cytotoxic T lymphocyte-associated protein 4(CTLA-4), programmed cell death protein 1(PD-1), lymphocyte activation gene 3(LAG-3), T cell immunoglobulin mucin-containing protein 3(TIM-3), B and T Lymphocyte Attenuator (BTLA), T cell immune receptor with Ig and ITIM domains (TIGIT), T cell activated V domain Ig suppressor (VISTA), adenosine A2a receptor (A2aR), killer immunoglobulin-like receptor (KIR), indoleamine 2, 3-dioxygenase (IDO), CD20, CD39, CD73, or any combination thereof.

In some aspects, the immunomodulator comprises an activator of a positive co-stimulatory molecule or an activator of a binding partner of a positive co-stimulatory molecule. In certain aspects, the positive co-stimulatory molecule is a member of the TNF receptor superfamily (e.g., CD120a, CD120b, CD18, OX40, CD40, Fas receptor, M68, CD27, CD30, 4-1BB, TRAILR1, TRAILR2, TRAILR3, TRAILR4, RANK, OCIF, TWEAK receptor, TACI, BAFF receptor, ATAR, CD271, CD269, AITR, TROY, CD358, TRAMP, and xedr). In some aspects, the activator of a positive co-stimulatory molecule is a member of the TNF superfamily (e.g., TNF α, TNF-C, OX40L, CD40L, FasL, LIGHT, TL1A, CD27L, Siva, CD153, 4-1BB ligand, TRAIL, RANKL, TWEAK, APRIL, BAFF, CAMLG, NGF, BDNF, NT-3, NT-4, GITR ligand, and EDA-2). In other aspects, the positive costimulatory molecule is a CD28 superfamily costimulatory molecule (e.g., ICOS or CD 28). In some aspects, the activator of a positive co-stimulatory molecule is ICOSL, CD80, or CD 86.

In some aspects, the immunomodulator comprises a cytokine or a binding partner for a cytokine. In certain aspects, the cytokine comprises IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21, or a combination thereof.

In some aspects, the immunomodulator comprises a protein that supports the intracellular interactions required for germinal center reactions. In certain aspects, the protein that supports the intracellular interactions required for germinal center reactions includes a Signaling Lymphocyte Activating Molecule (SLAM) family member or a SLAM-associated protein (SAP). In some aspects, the SLAM family member comprises SLAM family member 1, CD48, CD229(Ly9), Ly108, 2B4, CD84, NTB-a, CRACC, BLAME, CD2F-10, or a combination thereof.

II.D.10 inhibitors of lysophosphatidic acid (LPA)

In some aspects, the payload comprises a lysophosphatidic acid (LPA) inhibitor, such as an LPA-1 inhibitor. In certain aspects, the LPA-1 inhibitor is linked (e.g., chemically linked via a maleimide moiety) to a scaffold moiety, e.g., a scaffold X protein or fragment thereof, on the outer surface of an EV (e.g., exosome) or on the luminal surface of an EV (e.g., exosome). In some aspects, the LPA-1 inhibitor is linked (e.g., chemically linked via a maleimide moiety) to a scaffold moiety, such as a scaffold Y protein or fragment thereof, on the luminal surface of an EV (e.g., exosome). In other aspects, the LPA-1 inhibitor is in the lumen of an EV (e.g., exosome).

LPA is a highly potent endogenous lipid mediator that protects and rescues cells from programmed cell death. LPA acts as an important mediator of fibrogenesis through the action of its high affinity LPA-1 receptor. Thus, LPA inhibitors may act as anti-fibrotic agents.

In some aspects, the LPA-1 inhibitor comprises AM095, which is a potent and orally bioavailable antagonist of LPA-1, IC for mouse or recombinant human LPA-1₅₀The values were 0.73 and 0.98. mu.M, respectively. In vitro, AM095 has been shown to inhibit LPA-1-induced chemotaxis, IC, of mouse LPA-1/CHO cells and human A2058 melanoma cells₅₀The values were 0.78. mu.M and 0.23. mu.M. In vivo, AM095 can dose-dependently block LPA-induced histamine release, ED in mice₅₀The value was 8.3 mg/kg. Furthermore, AM095 has been found to significantly reduce BALF collagen and protein, ED, in the lung₅₀The value was 10 mg/kg. AM095 was also shown to reduce bleomycin-induced macrophage and lymphocyte infiltration in mice. See Swaney et al (2018) mol. Can. Res.16:1601-1613, which is incorporated herein by reference in its entirety.

In some aspects, the LPA-1 inhibitor comprises AM152 (also known as BMS-986020). AM152 is a high affinity LPA-1 antagonist, inhibiting bile acid and phospholipid transporters, and has activity against BSEP, MRP4 and IC of MDR3₅₀4.8. mu.M, 6.2. mu.M and 7.5. mu.M, respectively. AM152 can be used for the treatment of Idiopathic Pulmonary Fibrosis (IPF). See Kihara et al (2015) exp.cell Res.333: 171-7; rosen et al (2017) European Respiratory Journal 50: PA 1038; and palm et al (2018) Chest 154:1061-1069, which are incorporated herein by reference in their entirety. The phase 2 study of AM152 (described in Palmer 2018) was terminated prematurely due to gallbladder toxicity and early signs of hepatotoxic liver transporters (2 specific transporters).

Additional disclosure related to EVs (e.g., exosomes) comprising LPA-1 inhibitors is provided elsewhere in the present disclosure (see, e.g., example 5).

II.D.11 NLRP3 inhibitors

In some aspects, the payload comprises an inflammasome inhibitor, such as an NLRP3 inhibitor. In certain aspects, the NLRP3 inhibitor is linked (e.g., chemically linked via a maleimide moiety) to a scaffold moiety, such as a scaffold X protein or fragment thereof, on the outer surface of an EV (e.g., exosome) or on the luminal surface of an EV (e.g., exosome). In some aspects, the NLRP3 inhibitor is linked (e.g., chemically linked via a maleimide moiety) to a scaffold moiety, such as a scaffold Y protein or fragment thereof, on the luminal surface of an EV (e.g., an exosome). In other aspects, the NLRP3 inhibitor is in the cavity of an EV (e.g., exosome).

NLRP3, also known as NALP3, NACHT or icilin, is a protein encoded by the NLRP3 gene in humans. NLRP3 is expressed primarily in macrophages and is a component of the inflamed bodies. NLRP3 senses pathogen-derived, environmental and host-derived factors and contributes to the formation of inflammasomes, complexes associated with many inflammatory diseases. The NLRP3 inflammasome is an innate immune sensor that activates caspase-1 and mediates processing and release of IL-1 β after assembly. Mouse models of a number of diseases have been shown to be ameliorated by the absence of NLRP3 inflammasome, including gout, type 2 diabetes, multiple sclerosis, alzheimer's disease, and atherosclerosis. NLRP3 inflammasome plays a role in the pathogenesis of gout and neuroinflammation, for example in protein misfolding diseases such as alzheimer's disease, parkinson's disease and prion disease. Liu-Bryan (2010) Immunology and Cell Biology 88: 20-23; heneka et al (2013) Nature 493: 674-678; shi et al (2015) Life Sciences 135: 9-14; levy et al (2015) Nature Medicine 21: 213-215.

In some aspects, the NLPR3 inhibitor is a diarylsulfonylurea-containing compound. In some aspects, the diarylsulfonylurea-containing compound is MCC950 or a derivative thereof.

MCC950(N- [ [ (1,2,3,5,6, 7-hexahydro-sym-indacen-4-yl) amino ] carbonyl ] -4- (1-hydroxy-1-methylethyl) -2-furansulfonamide), also known as CP-456773, is a potent and selective inhibitor of NLRP3 (protein 3 containing NOD-like receptor (NLR) pyrin domain) inflammasome. MCC950 blocks the release of IL-1 β induced by NLRP3 activators such as ATP, MSU and nigericin by preventing oligomerization of the inflammatory body adapter protein ASC (CARD-containing apoptosis-related speck-like protein). Coll et al (2015) Nature Med.21: 248-. MCC950 blocks the release of IL-1 β in macrophages, which are primed by LPS and activated by ATP or nigericin with an IC50 of about 7.5 nM. While MCC950 would prevent NLRP 3-induced release of IL-1 β, MCC950 would not inhibit NLRC4, AIM2 or NLRP1 inflammasome. In addition, MCC950 does not inhibit TLR2 signaling or initiation of NLRP 3.

MCC950 is active in vivo, thereby blocking IL-1 β production and increasing survival in a mouse model of multiple sclerosis. MCC950 also inhibits NLRP 3-induced IL-1 β production in a myocardial infarction model. van Hout et al (2015) eur. MCC950 is also active in ex vivo samples from individuals with mookler-wells syndrome. MCC950 is a potential therapeutic agent for treating NLRP 3-related syndromes, including autoinflammatory and autoimmune diseases.

II.E biodistribution modulators

In some aspects, the EV (e.g., exosomes) comprise a biodistribution modulator. As used herein, the term "biodistribution modulator" refers to an agent (i.e., payload) that can modulate the distribution of extracellular vesicles (e.g., exosomes, nanovesicles) in vivo or in vitro (e.g., in mixed cultures of different varieties of cells). In some aspects, the term "targeting moiety" may be used interchangeably with the term biodistribution modulator. In some aspects, the targeting moiety alters the tropism ("tropism moiety") of an EV (e.g., exosome). As used herein, the term "tropism moiety" refers to a targeting moiety that when expressed on an EV (e.g., exosome) alters and/or enhances the natural motility of the EV. For example, in some aspects, the tropism moiety may promote uptake of EV by a particular cell, tissue or organ. Non-limiting examples of tropism moieties that can be used with the present disclosure include those that can bind to markers specifically expressed on dendritic cells (e.g., Clec9A or DEC205) or T cells (e.g., CD 3). The term "targeting moiety" as used herein includes a tropism moiety, unless otherwise indicated.

In some aspects, an EV (e.g., exosome) comprises a targeting moiety, i.e., a bioactive molecule that directs an EV (e.g., exosome) of the present disclosure to a particular cell type or tissue, which comprises a target (e.g., a target protein, such as a receptor), wherein another payload (e.g., another bioactive molecule) may have a therapeutic, prophylactic, or diagnostic effect. In certain aspects, the targeting moiety is linked (e.g., chemically linked via a maleimide moiety) to a scaffold moiety, such as a scaffold X protein or fragment thereof, on the outer surface of the EV (e.g., exosome).

In some aspects, the targeting moiety is an exogenous targeting moiety, such as an antibody or antigen binding portion thereof, a protein or peptide that specifically binds to a protein (e.g., a receptor) present on the surface of a target cell or tissue.

In some aspects, the targeting moiety specifically binds to a marker of a dendritic cell. In certain aspects, the marker is present only on dendritic cells. In some aspects, the dendritic cells comprise plasmacytoid dendritic cells (pdcs), bone marrow/conventional dendritic cell 1 (cdcs 1), bone marrow/conventional dendritic cell 2 (cdcs 2), or any combination thereof. In certain aspects, the dendritic cell is cDC 1. In other aspects, the marker comprises a C-type lectin domain family 9 member a (Clec9a) protein, dendritic cell specific intercellular adhesion molecule-3-capture non-integrin (DC-SIGN), CD207, CD40, Clec6, Dendritic Cell Immunoreceptor (DCIR), DEC-205, lectin-like oxidized low density lipoprotein receptor-1 (LOX-1), MARCO, Clec12a, DC-asialoglycoprotein receptor (DC-ASGPR), DC immunoreceptor 2(DCIR2), Dectin-1, Macrophage Mannose Receptor (MMR), BDCA-1(CD303, Clec4C), Dectin-2, Bst-2(CD317), or any combination thereof. In certain aspects, the marker is Clec9a protein.

In some aspects, an EV (e.g., exosome) of the present disclosure may comprise a tissue-or cell-specific ligand, i.e., a tropism moiety, that increases the tropism of the EV (e.g., exosome) to a particular tissue or cell. Thus, in some aspects, delivery of EVs (e.g., exosomes) to a particular tissue or cell type may be improved by linking a cell type-directed, tropism moiety (e.g., an immunoaffinity ligand that targets an antigen present on the surface of a certain neural cell type) to the EV (e.g., exosomes). In certain aspects, a tropism moiety is linked (e.g., chemically linked via a maleimide moiety) to a scaffold moiety, such as a scaffold X protein or fragment thereof, on the outer surface of an EV (e.g., exosome).

Tropism may be further improved by attaching anti-phagocytic signals (e.g., CD47 and/or CD24), half-life extending moieties (e.g., albumin or PEG), or any combination thereof to the outer surface of an EV (e.g., exosomes) of the present disclosure. In certain aspects, an anti-phagocytic signal is linked (e.g., chemically linked via a maleimide moiety) to a scaffold moiety, such as a scaffold X protein or fragment thereof, on the outer surface of an EV (e.g., exosome).

Pharmacokinetics, biodistribution, particularly tropism and retention in the desired tissue or anatomical location may also be achieved by selecting an appropriate route of administration (e.g., intrathecal or intraocular administration to improve tropism for the central nervous system).

In some aspects, when tropism for the central nervous system is desired, an EV (e.g., exosome) of the present disclosure may comprise a tissue-or cell-specific target ligand that increases the tropism of the EV (e.g., exosome) for a particular central nervous system tissue or cell. In some aspects, the cell is a glial cell. In some aspects, the glial cell is an oligodendrocyte, astrocyte, ependymal cell, microglial cell, schwann cell, satellite glial cell, olfactory ensheathing cell, or a combination thereof. In some aspects, the cell is a neural stem cell. In some aspects, a cell-specific target ligand that increases EV (e.g., exosome) tropism for schwann cells binds to schwann cell surface markers, such as Myelin Basic Protein (MBP), myelin protein zero (P0), P75NTR, NCAM, PMP22, and any combination thereof. In some aspects, the cell-specific tropism moiety comprises an antibody or antigen-binding portion thereof, an aptamer, or an agonist or antagonist of a receptor expressed on the surface of a schwann cell.

In principle, an EV (e.g., exosome) of the present disclosure comprising at least one tropism moiety that can direct the EV (e.g., exosome) to a particular target cell or tissue (e.g., schwann cell in peripheral nerve) can be administered using any suitable method of administration known in the art (e.g., intravenous injection or infusion) because the presence of the tropism moiety (alone or in combination with the presence of an anti-phagocytic signal and the use of a particular route of administration) will induce the tropism of the EV (e.g., exosome) towards the desired target cell or tissue.

In some aspects, a targeting moiety and/or a tropism moiety disclosed herein may be attached to an EV of the present disclosure (e.g., an exosome) via a scaffold moiety (e.g., a scaffold X protein moiety or fragment thereof, or a lipid moiety), wherein the targeting and/or tropism moiety is chemically attached to the scaffold moiety via a maleimide moiety and optionally one or more linkers (e.g., cleavable linkers).

II.F EV (e.g., exosome)

EVs (e.g., exosomes) of the present disclosure may have a diameter between about 20 and about 300 nm. In certain aspects, EVs (e.g., exosomes) of the present disclosure have between about 20 and about 290nm, between about 20 and about 280nm, between about 20 and about 270nm, between about 20 and about 260nm, between about 20 and about 250nm, between about 20 and about 240nm, between about 20 and about 230nm, between about 20 and about 220nm, between about 20 and about 210nm, between about 20 and about 200nm, between about 20 and about 190nm, between about 20 and about 180nm, between about 20 and about 170nm, between about 20 and about 160nm, between about 20 and about 150nm, between about 20 and about 140nm, between about 20 and about 130nm, between about 20 and about 120nm, between about 20 and about 110nm, between about 20 and about 100nm, between about 20 and about 90nm, between about 20 and about 80nm, between about 20 and about 70nm, between about 20 and about 60nm, Between about 20 and about 50nm, between about 20 and about 40nm, between about 20 and about 30nm, between about 30 and about 300nm, between about 30 and about 290nm, between about 30 and about 280nm, between about 30 and about 270nm, between about 30 and about 260nm, between about 30 and about 250nm, between about 30 and about 240nm, between about 30 and about 230nm, between about 30 and about 220nm, between about 30 and about 210nm, between about 30 and about 200nm, between about 30 and about 190nm, between about 30 and about 180nm, between about 30 and about 170nm, between about 30 and about 160nm, between about 30 and about 150nm, between about 30 and about 140nm, between about 30 and about 130nm, between about 30 and about 120nm, between about 30 and about 110nm, between about 30 and about 100nm, between about 30 and about 90nm, between about 30 and about 80nm, Between about 30 and about 70nm, between about 30 and about 60nm, between about 30 and about 50nm, between about 30 and about 40nm, between about 40 and about 300nm, between about 40 and about 290nm, between about 40 and about 280nm, between about 40 and about 270nm, between about 40 and about 260nm, between about 40 and about 250nm, between about 40 and about 240nm, between about 40 and about 230nm, between about 40 and about 220nm, between about 40 and about 210nm, between about 40 and about 200nm, between about 40 and about 190nm, between about 40 and about 180nm, between about 40 and about 170nm, between about 40 and about 160nm, between about 40 and about 150nm, between about 40 and about 140nm, between about 40 and about 130nm, between about 40 and about 120nm, between about 40 and about 110nm, between about 40 and about 100nm, between about 40 and about 90nm, between about 40 and about 100nm, Between about 40 and about 80nm, between about 40 and about 70nm, between about 40 and about 60nm, between about 40 and about 50nm, between about 50 and about 300nm, between about 50 and about 290nm, between about 50 and about 280nm, between about 50 and about 270nm, between about 50 and about 260nm, between about 50 and about 250nm, between about 50 and about 240nm, between about 50 and about 230nm, between about 50 and about 220nm, between about 50 and about 210nm, between about 50 and about 200nm, between about 50 and about 190nm, between about 50 and about 180nm, between about 50 and about 170nm, between about 50 and about 160nm, between about 50 and about 150nm, between about 50 and about 140nm, between about 50 and about 130nm, between about 50 and about 120nm, between about 50 and about 110nm, between about 50 and about 100nm, between about 50 and about 90nm, Between about 50 and about 80nm, between about 50 and about 70nm, between about 50 and about 60nm, between about 60 and about 300nm, between about 60 and about 290nm, between about 60 and about 280nm, between about 60 and about 270nm, between about 60 and about 260nm, between about 60 and about 250nm, between about 60 and about 240nm, between about 60 and about 230nm, between about 60 and about 220nm, between about 60 and about 210nm, between about 60 and about 200nm, between about 60 and about 190nm, between about 60 and about 180nm, between about 60 and about 170nm, between about 60 and about 160nm, between about 60 and about 150nm, between about 60 and about 140nm, between about 60 and about 130nm, between about 60 and about 120nm, between about 60 and about 110nm, between about 60 and about 100nm, between about 60 and about 90nm, between about 60 and about 60nm, between about 60 and about 190nm, Between about 60 and about 70nm, between about 70 and about 300nm, between about 70 and about 290nm, between about 70 and about 280nm, between about 70 and about 270nm, between about 70 and about 260nm, between about 70 and about 250nm, between about 70 and about 240nm, between about 70 and about 230nm, between about 70 and about 220nm, between about 70 and about 210nm, between about 70 and about 200nm, between about 70 and about 190nm, between about 70 and about 180nm, between about 70 and about 170nm, between about 70 and about 160nm, between about 70 and about 150nm, between about 70 and about 140nm, between about 70 and about 130nm, between about 70 and about 120nm, between about 70 and about 110nm, between about 70 and about 100nm, between about 70 and about 90nm, between about 70 and about 80nm, between about 80 and about 300nm, between about 70 and about 290nm, Between about 80 and about 280nm, between about 80 and about 270nm, between about 80 and about 260nm, between about 80 and about 250nm, between about 80 and about 240nm, between about 80 and about 230nm, between about 80 and about 220nm, between about 80 and about 210nm, between about 80 and about 200nm, between about 80 and about 190nm, between about 80 and about 180nm, between about 80 and about 170nm, between about 80 and about 160nm, between about 80 and about 150nm, between about 80 and about 140nm, between about 80 and about 130nm, between about 80 and about 120nm, between about 80 and about 110nm, between about 80 and about 100nm, between about 80 and about 90nm, between about 90 and about 300nm, between about 90 and about 290nm, between about 90 and about 280nm, between about 90 and about 270nm, between about 90 and about 90nm, between about 90 and about 250nm, between about 80 and about 250nm, A diameter between about 90 and about 240nm, between about 90 and about 230nm, between about 90 and about 220nm, between about 90 and about 210nm, between about 90 and about 200nm, between about 90 and about 190nm, between about 90 and about 180nm, between about 90 and about 170nm, between about 90 and about 160nm, between about 90 and about 150nm, between about 90 and about 140nm, between about 90 and about 130nm, between about 90 and about 120nm, between about 90 and about 110nm, between about 90 and about 100nm, between about 100 and about 300nm, between about 110 and about 290nm, between about 120 and about 280nm, between about 130 and about 270nm, between about 140 and about 260nm, between about 150 and about 250nm, between about 160 and about 240nm, between about 170 and about 230nm, between about 180 and about 220nm, or between about 190 nm. The size of an EV (e.g., exosome) described herein can be measured according to methods known in the art. The EVs of the present disclosure include exosomes, microvesicles, apoptotic bodies, or any combination thereof. In some aspects, the EVs of the present disclosure comprise an exosome and/or microvesicle population.

An EV (e.g., exosome) of the present disclosure comprises a lipid membrane ("exosome membrane" or "EV membrane") comprising an inner surface (lumen surface) and an outer surface (e.g., extracellular surface). The inner surface faces the inner core of the EV (e.g., exosome), i.e., the lumen of the EV. In certain aspects, the outer surface may be in contact with the membrane/cytoplasm of an endosome, multivesicular body, or producer cell.

In some aspects, EV (e.g., exosome) membranes comprise a lipid membrane, such as a lipid bilayer. In some aspects, EV (e.g., exosome) membranes comprise lipids and fatty acids. In some aspects, EV (e.g., exosome) membranes comprise lipids including phospholipids, glycolipids, fatty acids, sphingolipids, phosphoglycerides, sterols, cholesterol, and phosphatidylserine. In some aspects, an EV (e.g., exosome) membrane comprises an inner leaflet and an outer leaflet. The composition of the inner and outer leaflets can be determined by a trans-bilayer distribution assay known in the art, see, e.g., Kuypers et al, Biohim biophysis Acta 1985819: 170.

In some aspects, the composition of the outer leaflet is between about 70% -90% choline phospholipid, between about 0% -15% acid phospholipid, and between about 5% -30% phosphatidylethanolamine. In some aspects, the composition of the inner leaflet is between about 15% -40% choline phospholipid, between about 10% -50% acid phospholipid, and between about 30% -60% phosphatidylethanolamine. In some aspects, the EV or exosome membrane comprises one or more polysaccharides, such as glycans. Glycans on the surface of EVs or exosomes may serve as a linker for the maleimide moiety or a linker linking the glycan and the maleimide moiety. Glycans may be present on one or more proteins on the surface of an EV (e.g., exosome), for example, a scaffold X, such as a PTGFRN polypeptide, or on the lipid membrane of an EV (e.g., exosome). The glycans can be modified to have a thiafucose, which can serve as a functional group that attaches the maleimide moiety to the glycan. In some aspects, scaffold X can be modified to express a large number of glycans to allow additional ligation on EVs (e.g., exosomes).

II.G. scaffold moiety

In some aspects, the bioactive molecule is attached to the outer surface or cavity surface of the EV (e.g., exosome). In some aspects, a bioactive molecule is attached (e.g., chemically attached via a maleimide moiety) to a scaffold moiety (e.g., scaffold X) on the outer surface or luminal surface of an EV (e.g., exosome). In some aspects, a bioactive molecule is linked (e.g., chemically linked via a maleimide moiety) to a scaffold moiety (e.g., a cholesterol moiety) on the outer surface or luminal surface of an EV (e.g., an exosome).

For example, the full length mature PTGRN contains 16 cysteines, i.e. it contains 16 sulfhydryl groups, of which 14 are located in the extracellular portion of the protein and 2 in its intracellular portion. PTGRN has 6 disulfide bonds, all extracellular. Thus, PTGRN has 2 extracellular and 2 intracellular sulfhydryl groups. Thus, in some aspects, the biologically active moiety can be chemically linked to the scaffold X protein (e.g., PTGRN or a fragment thereof) via the maleimide moiety by a reaction between a maleimide reactive group present on the biologically active moiety and one of the thiols present on the scaffold X protein (e.g., PTGRN or a fragment thereof). In contrast, a scaffold X protein comprising a maleimide reactive group (e.g., PTGRN or a fragment thereof) is introduced by a reaction between a bifunctional reagent such as SMCC (succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate) and a lysine side chain of the scaffold X protein. The scaffold X protein may react with sulfhydryl groups present in a biologically active molecule.

In certain aspects, one or more moieties can be introduced into an EV (e.g., exosomes) by transfection. In some aspects, one or more moieties may be introduced into an EV (e.g., exosome) using synthetic macromolecules such as cationic lipids and polymers (Papapetrou et al, Gene Therapy 12: S118-S130 (2005)). In certain aspects, chemicals such as calcium phosphate, cyclodextrins, or polyaromatics may be used to introduce one or more moieties into an EV (e.g., exosomes).

In some aspects, one or more scaffold moieties can be CD47, CD55, CD49, CD40, CD133, CD59, glypican-1, CD9, CD63, CD81, integrins, selectins, lectins, cadherins, other similar polypeptides known to those of skill in the art, or any combination thereof.

In other aspects, one or more scaffold moieties are expressed in the membrane of an EV (e.g., exosome) by recombinantly expressing the scaffold moiety in a producer cell. EVs (e.g., exosomes) obtained from producer cells may be further modified to be conjugated with a maleimide moiety or linker. In other aspects, the scaffold moiety (e.g., scaffold X) is deglycosylated. In some aspects, the scaffold moiety (e.g., scaffold X) is highly glycosylated, e.g., higher than naturally occurring scaffold X under the same conditions.

G.1 transmembrane scaffold moiety (e.g., scaffold X)

Various modifications or fragments of the scaffold moiety may be used in aspects of the disclosure. For example, scaffold moieties modified to have enhanced affinity for binding agents can be used to generate surface-engineered EVs (e.g., exosomes) that can be purified using binding agents. Scaffold moieties modified to more effectively target EV (e.g., exosomes) and/or membrane may be used. Scaffold moieties modified to include the smallest fragment required to specifically and efficiently target EV (e.g., exosome) membranes may also be used. In some aspects, a scaffold moiety can be attached to a maleimide moiety described herein. In other aspects, the scaffold moiety is not attached to a maleimide moiety.

The scaffold moiety can be engineered synthetically or recombinantly, e.g., expressed as a fusion protein, e.g., a fusion protein of scaffold X with another moiety that can react with a maleimide on another molecule (e.g., a protein linker, a protein sequence comprising a reactive group (e.g., a thiol group), or a combination thereof). For example, a fusion protein may comprise a scaffold moiety disclosed herein (e.g., scaffold X, e.g., PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3a2, ATP transporter, or a fragment or variant thereof) linked to another moiety. In the case of a fusion protein, the second moiety may be a natural peptide, a recombinant peptide, a synthetic peptide, or any combination thereof. In other aspects, the scaffold moiety may be CD9, CD63, CD81, PDGFR, GPI protein, lactadherin, LAMP2, or LAMP2B, or any combination thereof. Non-limiting examples of other stent portions that may be used in the present disclosure include: aminopeptidase N (CD 13); enkephalinase, also known as Membrane Metalloendopeptidase (MME); ectonucleotide pyrophosphatase/phosphodiesterase family member 1(ENPP 1); neuropilin-1(NRP 1); or any combination thereof.

In some aspects, the fusion molecule may comprise a scaffold protein disclosed herein (e.g., PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3a2, ATP transporter, or a fragment or variant thereof) linked (e.g., chemically linked via a maleimide moiety) to a biologically active molecule, either directly or through an intermediate (e.g., a chemically inducible dimer, antigen binding domain, or receptor). In some aspects, the fusion molecule can be chemically linked to, for example, a targeting moiety or to a tropism moiety via a maleimide moiety.

In some aspects, the surface (e.g., scaffold X) engineered EVs (e.g., exosomes) described herein exhibit superior characteristics compared to EVs (e.g., exosomes) known in the art. For example, a surface (e.g., scaffold X) engineered comprises a modified protein that is more highly enriched on the outer surface or luminal surface of an EV (e.g., exosome) compared to a naturally occurring EV (e.g., exosome) or an EV (e.g., exosome) produced using a conventional EV (e.g., exosome) protein. Furthermore, surface (e.g., scaffold X) engineered EVs (e.g., exosomes) of the present disclosure may have greater, more specific, or more controllable biological activity compared to naturally occurring EVs (e.g., exosomes) or EVs (e.g., exosomes) produced using conventional EV (e.g., exosome) proteins.

In some aspects, the scaffold moiety (e.g., scaffold X protein) comprises a prostaglandin F2 receptor negative modulator (PTGFRN polypeptide). PTGFRN polypeptide may also be referred to as CD9 partner 1(CD9P-1), Glu-Trp-Ile EWI motif-containing protein F (EWI-F), prostaglandin F2-alpha receptor regulatory protein, prostaglandin F2-alpha receptor related protein, or CD 315. The full-length amino acid sequence of the human PTGFRN polypeptide (Uniprot accession Q9P2B2) is shown in Table 2 as SEQ ID NO: 1. The PTGFRN polypeptide comprises a signal peptide (amino acids 1 to 25 of SEQ ID NO: 1), an extracellular domain (amino acids 26 to 832 of SEQ ID NO: 1), a transmembrane domain (amino acids 833 to 853 of SEQ ID NO: 1) and a cytoplasmic domain (amino acids 854 to 879 of SEQ ID NO: 1). The mature PTGFRN polypeptide consists of SEQ ID NO. 1 without signal peptide, i.e. amino acids 26 to 879 of SEQ ID NO. 1. In some aspects, a PTGFRN polypeptide fragment useful in the present disclosure comprises a transmembrane domain of a PTGFRN polypeptide. In other aspects, a fragment of a PTGFRN polypeptide useful in the present disclosure comprises the transmembrane domain of a PTGFRN polypeptide and (i) comprises at least about five, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150 amino acids at the N-terminus of the transmembrane domain, (ii) comprises at least about five, at least about 10, at least about 15, at least about 20, or at least about 25 amino acids at the C-terminus of the transmembrane domain, or both (i) and (ii).

In some aspects, a fragment of a PTGFRN polypeptide lacks one or more functional or structural domains, such as IgV.

In other aspects, the scaffold moiety (e.g., scaffold X) comprises the amino acid sequence of SEQ ID NO:2, except for one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, or seven amino acid mutations. The mutation may be a substitution, insertion, deletion or any combination thereof. In some aspects, the scaffold moiety (e.g., scaffold X) comprises the amino acid sequence of SEQ ID NO:2 and comprises 1 amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, ten amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or more at the N-terminus and/or C-terminus of SEQ ID NO: 2.

In other aspects, the scaffold moiety (e.g., scaffold X) comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to amino acids 26 to 879 of SEQ ID No. 1, amino acids 833 to 853 of SEQ ID No. 1, SEQ ID No. 2, or SEQ ID No. 1. In other aspects, the scaffold X comprises amino acids 26 to 879 of SEQ ID No. 1, amino acids 833 to 853 of SEQ ID No. 1, SEQ ID No. 2, or the amino acid sequence of SEQ ID No. 1, but has one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, or seven amino acid mutations. The mutation may be a substitution, insertion, deletion or any combination thereof.

In some aspects, the scaffold moiety (e.g., scaffold X) comprises SEQ ID NO:1, amino acids 26 to 879 of SEQ ID NO:1, amino acids 833 to 853 of SEQ ID NO:2 or SEQ ID NO:1, or a pharmaceutically acceptable salt thereof, and in SEQ ID NO:1, amino acids 26 to 879 of SEQ ID NO:1, amino acids 833 to 853 of SEQ ID NO:2 or SEQ ID NO:1, 1 amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, ten amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or longer at the N-terminus and/or C-terminus of the amino acid sequence of 1.

In other aspects, a scaffold moiety (e.g., scaffold X) comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:186, 187, 188, 189, 190, or 191. In other aspects, the scaffold moiety (e.g., scaffold X) comprises the amino acid sequence of SEQ ID NO:186, 187, 188, 189, 190, or 191, in addition to one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, or seven amino acid mutations. The mutation may be a substitution, insertion, deletion or any combination thereof. In some aspects, the scaffold moiety (e.g., scaffold X) comprises the amino acid sequence of SEQ ID NO:186, 187, 188, 189, 190, or 191 and comprises 1 amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, ten amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or more at the N-terminus and/or C-terminus of SEQ ID NO:186, 187, 188, 189, 190, or 191.

TABLE 2 exemplary scaffold protein sequences

In other embodiments, a scaffold moiety (e.g., scaffold X) comprises a BSG protein, an IGSF8 protein, an IGSF3 protein, an ITGB1 protein, a SLC3a2 protein, an ITGA4 protein, an ATP1a1 protein, an ATP1a2 protein, an ATP1A3 protein, an ATP1a4 protein, an ATP1a5 protein, an ATP2B 5 protein, or an IGSF 5 protein comprising at least about 100%, at least about 75%, at least about 75%, at least about 75%, or about at least about the same amino acid as the corresponding mature BSG protein as a mature BSG protein, at least about a mature. In some aspects, the BSG protein, IGSF8 protein, IGSF3 protein, ITGB1 protein, SLC3a2 protein, ITGA4 protein, ATP1a1 protein, ATP1a2 protein, ATP1A3 protein, ATP1a4 protein, ATP1a5 protein, ATP2B1 protein, ATP2B2 protein, ATP2B3 protein, ATP2B4 protein, or IGSF2 protein lacks one or more functional or structural domains, such as IgV.

Non-limiting examples of other scaffold X proteins can be found in US patent No. US 10,195,290B1 issued on 5.2.2019, which is incorporated by reference in its entirety, ATP transporter: ATP1a1, ATP1a2, ATP1A3, ATP1a4, ATP1B3, ATP2B1, ATP2B2, and ATP2B4), CD9, CD63, CD81, PDGFR, GPI protein, lectin, LAMP2, and LAMP 2B.

In some aspects, the scaffold moiety (e.g., scaffold X) comprises a base immunoglobulin (BSG protein). BSG protein is also known as 5F7, collagenase stimulating factor, extracellular matrix metalloproteinase inducer (EMMPRIN), leukocyte activation antigen M6, OK blood group antigen, tumor cell derived collagenase stimulating factor (TCSF), or CD 147. The Uniprot number of the human BSG protein is P35613. The signal peptide of the BSG protein is amino acids 1 to 21 of SEQ ID NO 3. Amino acids 138-323 of SEQ ID NO:3 are the extracellular domain, amino acids 324 to 344 of SEQ ID NO:3 are the transmembrane domain, and amino acids 345 to 385 of SEQ ID NO:3 are the cytoplasmic domain of BSG.

In other aspects, the scaffold moiety (e.g., scaffold X) comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 22 to 385 of the human BSG protein (SEQ ID NO: 3). In some aspects, a fragment of a Basigin polypeptide lacks one or more functional or structural domains, such as igvs, e.g., amino acids 221 through 315 of a human BSG protein.

In other aspects, a scaffold moiety (e.g., scaffold X) comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to SEQ ID NOs 193, 194, or 195. In other aspects, the scaffold moiety (e.g., scaffold X) comprises the amino acid sequence of SEQ ID NO:193, 194, or 195, except for one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, or seven amino acid mutations. The mutation may be a substitution, insertion, deletion or any combination thereof. In some aspects, the scaffold moiety (e.g., scaffold X) comprises the amino acid sequence of SEQ ID NO:193, 194, or 195 and comprises 1 amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, ten amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or more at the N-terminus and/or C-terminus of SEQ ID NO:193, 194, or 195.

In some aspects, the scaffold moiety (e.g., scaffold X) comprises immunoglobulin superfamily member 8(IgSF8 or IgSF8 protein), also referred to as CD81 partner 3, Glu-Trp-Ile EWI motif-containing protein 2(EWI-2), keratinocyte-associated transmembrane protein 4(KCT-4), LIR-D1, prostaglandin regulatory-like Protein (PGRL), or CD 316. The full-length human IGSF8 protein has accession number Q969P0 in Uniprot, shown herein as SEQ ID NO 4. The human IGSF8 protein has a signal peptide (amino acids 1 to 27 of the human IGSF8 protein; SEQ ID NO:4), an extracellular domain (amino acids 28 to 579 of the human IGSF8 protein; SEQ ID NO:4), a transmembrane domain (amino acids 580 to 600 of the human IGSF8 protein; SEQ ID NO:4), and a cytoplasmic domain (amino acids 601 to 613 of the human IGSF8 protein; SEQ ID NO: 4).

In other aspects, a scaffold moiety (e.g., scaffold X) comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 28 to 613(SEQ ID NO:4) of a human IGSF8 protein. In some aspects, IGSF8 proteins lack one or more functional or structural domains, such as igvs. In other aspects, the scaffold moiety (e.g., scaffold X) comprises the amino acid sequence of a human IGSF8 protein (SEQ ID NO:4) in addition to one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, or seven amino acid mutations. The mutation may be a substitution, insertion, deletion or any combination thereof. In some aspects, the scaffold moiety (e.g., scaffold X) comprises the amino acid sequence of a human IGSF8 protein (SEQ ID NO:4) and comprises 1 amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, ten amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or more at the N-terminus and/or C-terminus of a human IGSF8 protein (SEQ ID NO: 4).

In some aspects, a scaffold moiety (e.g., scaffold X) comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to SEQ ID NOs 197, 198, 199, or 200. In other aspects, the scaffold moiety (e.g., scaffold X) comprises the amino acid sequence of SEQ ID NO:197, 198, 199, or 200, in addition to one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, or seven amino acid mutations. The mutation may be a substitution, insertion, deletion or any combination thereof. In some aspects, the scaffold moiety (e.g., scaffold X) comprises the amino acid sequence of SEQ ID NO:197, 198, 199, or 200 and comprises 1 amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, ten amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or more at the N-terminus and/or C-terminus of SEQ ID NO:197, 198, 199, or 200.

In some aspects, a scaffold moiety (e.g., scaffold X) for use in the present disclosure comprises immunoglobulin superfamily member 3(IgSF3 or IGSF3 protein), also referred to as Glu-Trp-Ile EWI motif-containing protein 3(EWI-3), and shown as the amino acid sequence of SEQ ID NO: 203. The human IGSF3 protein has a signal peptide (amino acids 1 to 19 of IGSF3 protein of SEQ ID NO:203), an extracellular domain (amino acids 20 to 1124 of IGSF3 protein of SEQ ID NO:203), a transmembrane domain (amino acids 1125 to 1145 of IGSF3 protein), and a cytoplasmic domain (amino acids 1146 to 1194 of IGSF3 protein of SEQ ID NO: 203).

In some aspects, a scaffold moiety (e.g., scaffold X) for use in the present disclosure comprises integrin beta-1 (ITGB1 protein), which is also known as fibronectin receptor subunit beta, glycoprotein IIa (GPIIA), VLA-4 subunit beta, or CD29, and is shown as the amino acid sequence of SEQ ID NO: 5. The human ITGB1 protein has a signal peptide (amino acids 1 to 20 of the human ITGB1 protein of SEQ ID NO: 5), an extracellular domain (amino acids 21 to 728 of the human ITGB1 protein of SEQ ID NO: 5), a transmembrane domain (amino acids 729 to 751 of the human ITGB1 protein of SEQ ID NO: 5), and a cytoplasmic domain (amino acids 752 to 798 of the human ITGB1 protein of SEQ ID NO: 5).

In other aspects, the scaffold moiety (e.g., scaffold X) comprises an ITGA4 protein comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the human ITGB1 protein of SEQ ID NO:6 (amino acids 1 to 33 of the human ITGB1 protein of SEQ ID NO: 6) without the signal peptide. In some aspects, the ITGA4 protein lacks one or more functional or structural domains, such as IgV.

In other aspects, the scaffold moiety (e.g., scaffold X) comprises a SLC3a2 protein comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the SLC3a2 protein of SEQ ID No. 7 without the signal peptide. In some aspects, the SLC3a2 protein lacks one or more functional or structural domains, such as IgV.

In other aspects, the scaffold moiety (e.g., scaffold X) comprises an ATP1a1 protein comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the ATP1a1 protein of SEQ ID No. 204 without the signal peptide. In some aspects, the ATP1a1 protein lacks one or more functional or structural domains, such as IgV.

In other aspects, the scaffold moiety (e.g., scaffold X) comprises an ATP1a2 protein comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the ATP1a2 protein of SEQ ID No. 205 without the signal peptide. In some aspects, the ATP1a2 protein lacks one or more functional or structural domains, such as IgV.

In other aspects, the scaffold moiety (e.g., scaffold X) comprises an ATP1A3 protein comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the ATP1A3 protein of SEQ ID No. 206 without the signal peptide. In some aspects, the ATP1a3 protein lacks one or more functional or structural domains, such as IgV.

In other aspects, the scaffold moiety (e.g., scaffold X) comprises an ATP1a4 protein comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the ATP1a4 protein of SEQ ID NO:207 without the signal peptide. In some aspects, the ATP1a4 protein lacks one or more functional or structural domains, such as IgV.

In other aspects, the scaffold moiety (e.g., scaffold X) comprises an ATP1B3 protein comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the ATP1B3 protein of SEQ ID NO:208 without the signal peptide. In some aspects, the ATP1B3 protein lacks one or more functional or structural domains, such as IgV.

In other aspects, the scaffold moiety (e.g., scaffold X) comprises an ATP2B1 protein comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the ATP2B1 protein of SEQ ID No. 209 without the signal peptide. In some aspects, the ATP2B1 protein lacks one or more functional or structural domains, such as IgV.

In other aspects, the scaffold moiety (e.g., scaffold X) comprises an ATP2B2 protein comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the ATP2B2 protein of SEQ ID NO:210 without the signal peptide. In some aspects, the ATP2B2 protein lacks one or more functional or structural domains, such as IgV.

In other aspects, the scaffold moiety (e.g., scaffold X) comprises an ATP2B3 protein comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the ATP2B3 protein of SEQ ID NO:211 without the signal peptide. In some aspects, the ATP2B3 protein lacks one or more functional or structural domains, such as IgV.

In other aspects, the scaffold moiety (e.g., scaffold X) comprises an ATP2B4 protein comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the ATP2B4 protein of SEQ ID No. 212 without the signal peptide. In some aspects, the ATP2B4 protein lacks one or more functional or structural domains, such as IgV.

In other aspects, a scaffold moiety (e.g., scaffold X) comprises an IGSF2 protein comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to IGSF2 protein without a signal peptide (SEQ ID NO: 202). In some aspects, IGSF2 proteins lack one or more functional or structural domains, such as igvs.

Non-limiting examples of other scaffold moieties (e.g., scaffold X protein) can be found in US patent No. US10195290B1 issued 2/5 in 2019, which is incorporated by reference in its entirety.

In some aspects, the sequence encodes a fragment of the scaffold moiety that lacks at least about 5, at least about 10, at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, or at least about 800 amino acids from the N-terminus of the native protein. In some aspects, the sequence encodes a fragment of the scaffold moiety that lacks at least about 5, at least about 10, at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, or at least about 800 amino acids from the C-terminus of the native protein. In some aspects, the sequence encodes a fragment of the scaffold moiety that lacks at least about 5, at least about 10, at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, or at least about 800 amino acids from the N-terminus and C-terminus of the native protein. In some aspects, the sequences encode fragments of the scaffold portion that lack one or more functional or structural domains of the native protein.

In some aspects, a scaffold moiety (e.g., scaffold X), such as a PTGFRN protein, is linked to one or more heterologous proteins. One or more heterologous proteins may be attached to the N-terminus of the scaffold moiety. One or more heterologous proteins may be attached to the C-terminus of the scaffold moiety. In some aspects, one or more heterologous proteins are linked to the N-terminus and C-terminus of the scaffold moiety. In some aspects, the heterologous protein is a mammalian protein. In some aspects, the heterologous protein is a human protein.

In some aspects, a scaffold moiety (e.g., scaffold X) may be used to simultaneously attach any moiety to the luminal and the abluminal surfaces of an EV (e.g., exosome). For example, PTGFRN polypeptides may be used to attach one or more biologically active molecules through a maleimide moiety to either indirectly or directly to the maleimide moiety or to a linker attached to the luminal and outer surfaces of an EV (e.g., exosomes). Thus, in certain aspects, the stent X may serve a dual purpose.

In other aspects, the EVs (e.g., exosomes) of the disclosure comprise a greater number of scaffold X proteins than naturally-occurring EVs (e.g., exosomes). In some aspects, EVs (e.g., exosomes) of the present disclosure comprise at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 110-fold, at least about 120-fold, at least about 130-fold, at least about 140-fold, at least about 150-fold, at least about 160-fold, at least about 170-fold, at least about 180-fold, at least about 190-fold, at least about 200-fold, at least about 210-fold, at least about 220-fold, at least about 230-fold, at least about 240-fold, at least about 250-fold, at least about 260-fold, at least about 270-fold greater number of scaffold X (e.g., PTGFRN polypeptide) than the naturally-occurring EVs (e.g., exosomes).

The number of scaffold moieties (e.g., scaffold X, such as PTGFRN polypeptides) on an EV (e.g., exosomes) of the present disclosure is at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2100, at least about 2200, at least about 2300, at least about 2400, at least about 2500, at least about 2600, at least about 2700, at least about 2800, at least about 2900, at least about 3000, at least about 4000, at least about 5000, at least about 6000, at least about 7000, at least about 8000, at least about 9000, or at least about 10000.

In some aspects, the number of scaffold moieties (e.g., scaffold X, such as PTGFRN polypeptide) on an EV (e.g., exosome) of the present disclosure is from about 100 to about 100,000, from about 200 to about 9000, from about 300 to about 9000, from about 400 to about 9000, from about 500 to about 9000, from about 600 to about 8000, from about 800 to about 8000, from about 900 to about 8000, from about 1000 to about 8000, from about 1100 to about 8000, from about 1200 to about 8000, from about 1300 to about 8000, from about 1400 to about 8000, from about 1500 to about 8000, from about 1600 to about 8000, from about 1700 to about 8000, from about 1800 to about 8000, from about 1900 to about 8000, from about 2000 to about 8000, from about 2100 to about 8000, from about 2200 to about 8000, from about 2300 to about 8000, from about 2500 to about 8000, from about 2600, from about 2700 to about 8000, from about 2800 to about 8000, from about 8000 to about 8000, from about 2200 to about 8000, from about 3000 to about 8000, from about 3000, From about 6000 to about 8000, from about 7000 to about 8000, from 7000 to about 9000 or from about 6000 to about 10000.

In some aspects, the number of scaffold moieties (e.g., scaffold X, such as a PTGFRN polypeptide) on an EV (e.g., exosome) of the present disclosure is from about 5000 to about 8000, e.g., about 5000, about 6000, about 7000, or about 8000. In some aspects, the number of scaffold moieties (e.g., scaffold X, such as a PTGFRN polypeptide) on an EV (e.g., exosome) of the present disclosure is from about 6000 to about 8000, e.g., about 6000, about 7000, or about 8000. In some aspects, the number of scaffold moieties (e.g., scaffold X, such as a PTGFRN polypeptide) on an EV (e.g., exosome) of the present disclosure is from about 4000 to about 9000, e.g., about 4000, about 5000, about 6000, about 7000, about 8000, or about 9000.

II.G.2 Cavity support part (e.g. support Y)

In some aspects, an EV (e.g., exosome) of the present disclosure includes an internal space (i.e., a cavity) that is different from the internal space of a naturally-occurring EV (e.g., exosome). For example, an EV (e.g., exosome) may be altered such that the composition on the luminal surface of the EV (e.g., exosome) has a protein, lipid, or glycan content that is different from the protein, lipid, or glycan content of the naturally-occurring EV (e.g., exosome).

In some aspects, engineered EVs (e.g., exosomes) may be produced from cells transformed with exogenous sequences encoding a scaffold moiety (e.g., an exosome protein, e.g., scaffold Y) or a modification or fragment of the scaffold moiety that alters the composition or content of the luminal surface of the exosome. Various modifications or fragments of EV (e.g., exosomes) proteins that can be expressed on the luminal surface of an EV (e.g., exosomes) can be used in aspects of the disclosure.

In some aspects, EV (e.g., exosome) proteins that can alter the luminal surface of an EV (e.g., exosome) include, but are not limited to, MARCKS proteins, MARCKSL1 proteins, BASP1 proteins, or any combination thereof. In some aspects, the scaffold moiety (e.g., scaffold Y) comprises brain acid soluble protein 1(BASP1 protein). The BASP1 protein is also known as 22kDa neuronal tissue-enriched acidic protein or neuronal axon membrane protein NAP-22. The full-length human BASP1 protein sequence (isoform 1) is shown in table 3. The isoforms produced by alternative splicing lack amino acids 88 to 141 (isoform 1) of the BASP1 protein in table 3.

TABLE 3 exemplary scaffold protein sequences

In some aspects, the scaffold moiety (e.g., scaffold Y) comprises a protein selected from the group consisting of: MARCKS, MARKSL1, BASP1, any functional fragment, variant or derivative thereof or any combination thereof. In some aspects, the scaffold moiety (e.g., scaffold Y) comprises a Src protein or fragment thereof. In some aspects, a scaffold moiety (e.g., scaffold Y) comprises a sequence disclosed in, for example, U.S. patent No. 9,611,481.

In some aspects, a scaffold moiety (e.g., scaffold Y) of the present disclosure comprises a MARCKS protein, or a fragment, variant, or derivative thereof. MARCKS protein (Uniprot accession number P29966) is also known as protein kinase C substrate, 80kDa protein, light chain. The full-length human MARCKS protein is 332 amino acids in length and comprises a calmodulin binding domain at amino acid residues 152-176. In some aspects, a scaffold moiety of the disclosure (e.g., scaffold Y) comprises a mature MARCKS protein (i.e., no N-terminal methionine). In some aspects, the scaffold moiety (e.g., scaffold Y) of the present disclosure is derived from a mature MARCKS protein, i.e., it is a fragment, variant, or derivative of a mature MARCKS protein, and thus it lacks the N-terminal protein present in the non-mature protein.

In some aspects, a scaffold moiety of the disclosure (e.g., scaffold Y) comprises MARCKSL1 protein (Uniprot accession number P49006), also known as MARCKS-like protein 1 and a macrophage myristoylated alanine-rich C kinase substrate. The full-length human MARCKSL1 protein was 195 amino acids in length. The MARCKSL1 protein has an effector domain at amino acid residues 87-110 that is involved in lipid binding and calmodulin binding. In some aspects, a scaffold moiety of the disclosure (e.g., scaffold Y) comprises a mature MARCKSL1 protein (i.e., no N-terminal methionine). In some aspects, the scaffold moiety of the disclosure (e.g., scaffold Y) is derived from the mature MARCKSL1 protein, i.e., it is a fragment, variant, or derivative of mature MARCKSL1, and thus it lacks the N-terminal protein present in the non-mature protein.

In some aspects, a scaffold moiety (e.g., scaffold Y) of the present disclosure comprises BASP1 protein (Uniprot accession number P80723), also known as 22kDa neuronal tissue-rich acidic protein or neuronal axon membrane protein NAP-22. The full-length human BASP1 protein sequence (isoform 1) was 227 amino acids in length. Isoforms produced by alternative splicing lack amino acids 88 to 141 of isoform 1. In some aspects, a scaffold moiety of the disclosure (e.g., scaffold Y) comprises a mature BASP1 protein (i.e., without the N-terminal methionine). In some aspects, the scaffold moiety (e.g., scaffold Y) of the present disclosure is derived from mature BASP1 protein, i.e., it is a fragment, variant, or derivative of mature BASP1, and thus it lacks the N-terminal protein present in the immature protein. The mature BASP1 protein sequence lacks the first Met of SEQ ID NO. 10 and therefore contains amino acids 2 through 227 of SEQ ID NO. 10.

In other aspects, scaffold moieties useful in the present disclosure (e.g., scaffold Y) comprise an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to amino acids 2 through 227 of SEQ ID NO:10, i.e., the mature form of BASP1 (i.e., without the N-terminal methionine amino acid present in SEQ ID NO: 10). In other aspects, a scaffold moiety (e.g., a scaffold X protein) comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a functional fragment of the mature form of SEQ ID No. 10(BASP1) (i.e., without the N-terminal methionine amino acid present in SEQ ID No. 10). In other aspects, a scaffold moiety (e.g., scaffold Y) useful in the present disclosure comprises the amino acid sequence of SEQ ID NO:10, in addition to one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, or seven amino acid mutations. The mutation may be a substitution, insertion, deletion or any combination thereof. In some aspects, a scaffold moiety (e.g., scaffold Y) useful in the present disclosure comprises the amino acid sequence of SEQ ID NO:10 and comprises 1 amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, ten amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or more at the N-terminus and/or C-terminus of SEQ ID NO: 10.

In other aspects, a scaffold moiety (e.g., scaffold Y) useful in the present disclosure comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the mature form of SEQ ID No. 9(MARCKSL1) (i.e., without the N-terminal methionine amino acid present in SEQ ID No. 9). In other aspects, the scaffold moiety (e.g., scaffold Y) comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to a functional fragment of the mature form of SEQ ID No. 9(MARCKSL1) (i.e., without the N-terminal methionine amino acid present in SEQ ID No. 9). In other aspects, a scaffold moiety (e.g., scaffold Y) useful in the present disclosure comprises the amino acid sequence of SEQ ID No. 9, in addition to one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, or seven amino acid mutations. The mutation may be a substitution, insertion, deletion or any combination thereof. In some aspects, a scaffold moiety (e.g., scaffold Y) useful in the present disclosure comprises the amino acid sequence of SEQ ID NO:9 and comprises 1 amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, ten amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or more at the N-terminus and/or C-terminus of SEQ ID NO: 9.

In other aspects, a scaffold moiety (e.g., scaffold Y) useful in the present disclosure comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the mature form of SEQ ID No. 8(MARCKS) (i.e., without the N-terminal methionine amino acid present in SEQ ID No. 8). In other aspects, the scaffold moiety (e.g., scaffold Y) comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to a functional fragment of the mature form of SEQ ID No. 8(MARCKS) (i.e., without the N-terminal methionine amino acid present in SEQ ID No. 8). In other aspects, a scaffold moiety (e.g., scaffold Y) useful in the present disclosure comprises the amino acid sequence of SEQ ID NO:8 in addition to one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, or seven amino acid mutations. The mutation may be a substitution, insertion, deletion or any combination thereof. In some aspects, a scaffold moiety (e.g., scaffold Y) useful in the present disclosure comprises the amino acid sequence of SEQ ID NO:8 and comprises 1 amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, ten amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or more at the N-terminus and/or C-terminus of SEQ ID NO: 8.

In certain aspects, any of the protein sequences of SEQ ID NOs: 1-109 disclosed in PCT/US2018/061679 are sufficient as scaffold Y (e.g., a scaffold moiety linked to a linker) of the present disclosure.

In certain aspects, a scaffold moiety (e.g., scaffold Y) useful in the present disclosure comprises a peptide having MGXKLSKKK (SEQ ID NO:224) or GXKLSKKK (SEQ ID NO:225), wherein X is alanine or any other amino acid. In some aspects, an EV (e.g., exosome) comprises a peptide having the sequence (M) (G) (pi) (xi) (Φ/pi) (S/a/G/N) (+) or (G) (pi) (xi) (S/a/G/N) (+) where each bracketed position represents an amino acid, and where pi is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), xi is any amino acid selected from the group consisting of (Asn, gin, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein the fifth position is not (+) and the sixth position is neither (+) nor (Asp or Glu). In other aspects, an EV (e.g., exosome) (e.g., engineered exosome) described herein comprises a peptide having the sequence (M) (G) (pi) (X) (Φ/pi) (+) or (G) (pi) (X) (Φ/pi) (+) where each bracketed position represents an amino acid, and where pi is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, Ile, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein the fifth position is not (+) and the sixth position is neither (+) nor (Asp or Glu). See Aasland et al, FEBS Letters 513(2002)141-144 for amino acid nomenclature.

In other aspects, a scaffold moiety (e.g., scaffold Y) comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to any of the sequences disclosed in US 10,195,290B1 published on 2/5 2019.

Stent-Y engineered exosomes described herein can be produced from cells transformed with any of the sequences shown in PCT/US2018/061679 (SEQ ID NOs: 4-109 of PCT/US 2018/061679).

In other aspects, the EVs (e.g., exosomes) of the disclosure comprise a greater number of scaffold Y proteins than naturally-occurring EVs (e.g., exosomes). In some aspects, an EV (e.g., exosome) of the disclosure comprises at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 110-fold, at least about 120-fold, at least about 130-fold, at least about 140-fold, at least about 150-fold, at least about 160-fold, at least about 170-fold, at least about 180-fold, at least about 190-fold, at least about 200-fold, at least about 210-fold, at least about 220-fold, at least about 230-fold, at least about 240-fold, at least about 250-fold, at least about 260-fold, at least about 270-fold greater amount of scaffold Y (e.g., BASP-1 polypeptide) than the naturally-occurring EV (e.g., exosome). The number of scaffold Y (e.g., BASP-1 polypeptide) on an EV (e.g., exosome) of the present disclosure is at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2100, at least about 2200, at least about 2300, at least about 2400, at least about 2500, at least about 2600, at least about 2700, at least about 2800, at least about 2900, at least about 3000, at least about 4000, at least about 5000, at least about 6000, at least about 7000, at least about 8000, at least about 9000, or at least about 10000. In some aspects, the number of scaffold Y (e.g., BASP-1 polypeptides) on an EV (e.g., exosome) of the present disclosure is from about 100 to about 100,000, from about 200 to about 9000, from about 300 to about 9000, from about 400 to about 9000, from about 500 to about 9000, from about 600 to about 8000, from about 800 to about 8000, from about 900 to about 8000, from about 1000 to about 8000, from about 1100 to about 8000, from about 1200 to about 8000, from about 1300 to about 8000, from about 1400 to about 8000, from about 1500 to about 8000, from about 1600 to about 8000, from about 1700 to about 8000, from about 1800 to about 8000, from about 1900 to about 8000, from about 2000 to about 8000, from about 2100 to about 8000, from about 2200 to about 8000, from about 2300 to about 8000, from about 2400 to about 8000, from about 2500 to about 8000, from about 2600, from about 2700 to about 8000, from about 5000 to about 8000, from about 3000 to about 8000, from about 3000, from about 8000, from about 3000 to about 8000, from about 8000, From about 7000 to about 8000, from 7000 to about 9000 or from about 6000 to about 10000. In some aspects, the number of scaffold Y (e.g., BASP-1 polypeptides) on an EV (e.g., exosome) of the present disclosure is from about 5000 to about 8000, e.g., about 5000, about 6000, about 7000, or about 8000. In some aspects, the number of scaffold Y (e.g., BASP-1 polypeptides) on an EV (e.g., exosome) of the present disclosure is from about 6000 to about 8000, e.g., about 6000, about 7000, or about 8000. In some aspects, the number of scaffold Y (e.g., BASP-1 polypeptides) on E V (e.g., exosomes) of the present disclosure is from about 4000 to about 9000, e.g., about 4000, about 5000, about 6000, about 7000, about 8000, about 9000.

In some aspects, a scaffold moiety (e.g., scaffold Y) useful in the present disclosure comprises an "N-terminal domain" (ND) and an "effector domain" (ED), wherein ND and/or ED are associated with the luminal surface of an EV (e.g., exosome). In some aspects, a scaffold moiety useful in the present disclosure (e.g., scaffold Y) comprises an intracellular domain, a transmembrane domain, and an extracellular domain; wherein the intracellular domain comprises an "N-terminal domain" (ND) and an "effector domain" (ED); wherein ND and/or ED are associated with the luminal surface of the EV (e.g., exosomes). As used herein, the term "associated with … …" refers to the interaction between the scaffold proteins of the present disclosure and the luminal surface of an EV (e.g., exosome), which does not involve covalent attachment to a membrane component. For example, scaffold moieties useful in the present disclosure may be associated with the cavity surface of an EV, for example, by a lipid (e.g., myristic acid) and/or a polylomain that electrostatically interacts with a negatively charged membrane phospholipid head. In other aspects, a scaffold moiety (e.g., scaffold Y) comprises an N-terminal domain (ND) and an Effector Domain (ED), wherein ND is associated with the luminal surface of the EV by ionic interaction and ED is associated with the luminal surface of the EV, wherein ED comprises, in order, at least two, at least three, at least four, at least five, at least six, or at least seven consecutive basic amino acids, e.g., lysine (Lys).

In other aspects, a scaffold moiety (e.g., scaffold Y) comprises an N-terminal domain (ND) and an Effector Domain (ED), wherein ND is associated with a luminal surface of an EV by ionic interaction and ED is associated with a luminal surface of an EV, wherein ED comprises, in order, at least two, at least three, at least four, at least five, at least six, or at least seven consecutive lysines (Lys).

In other aspects, the ED further comprises one or more low complexity regions, such as a PEST motif. PEST sequences are peptide sequences rich in proline (P), glutamic acid (E), serine (S) and threonine (T). In some aspects, ED also comprises negatively charged residues (e.g., Glu) and many Ser and Thr that undergo transient phosphorylation (thus, both add negative charge to regions outside the ED).

In some aspects, the ND is associated with the luminal surface of the EV (e.g., exosome) by lipidation, e.g., by myristoylation. In some aspects, ND has Gly at the N-terminus. In some aspects, the N-terminal Gly is myristoylated.

In some aspects, the ED associates with the luminal surface of the EV (e.g., exosomes) through ionic interactions. In some aspects, the ED associates with the cavity surface of the EV (e.g., exosomes) through electrostatic interactions, particularly attractive electrostatic interactions.

In some aspects, the ED comprises (i) a basic amino acid (e.g., lysine), or (ii) two or more basic amino acids (e.g., lysine) adjacent to each other in the polypeptide sequence. In some aspects, the basic amino acid is lysine (Lys; K), arginine (Arg, R), or histidine (His, H). In some aspects, the basic amino acid is (Lys) n, wherein n is an integer between 1 and 10.

In some aspects, the ED comprises (i) a lysine repeat in the ED or (ii) a lysine repeat having an ND, e.g., a C-terminal K in ND and an N-terminal K in ED, wherein ND and ED are directly linked, i.e., linked by a peptide bond. In some aspects, the minimum number of amino acids capable of linking a heterologous moiety (e.g., a biologically active molecule) in the cavity of an EV (e.g., an exosome) is, for example, about seven to about 15, about seven to about 14, about seven to about 13, about seven to about 12, about seven to about 11, about seven to about 10, about seven to about 9, or about seven to about 8 amino acid fragments.

In other aspects, if the N-terminus of ED is directly linked to the C-terminal lysine of ND, i.e., the lysine is at the N-terminus of ED and fused to the C-terminal lysine of ND, then ED comprises at least lysine and ND comprises C-terminal lysine ED. In other aspects, when the N-terminus of ED is linked to the C-terminus of ND by a linker (e.g., one or more amino acids), the ED comprises at least two lysines, at least three lysines, at least four lysines, at least five lysines, at least six lysines, or at least seven lysines. In some aspects, ED comprises at least two consecutive lysines (Lys) in sequence.

In some aspects, ED comprises K, KK, KKK, KKKKKK (SEQ ID NO:11), KKKKKKK (SEQ ID NO:12), R, RR, RRR, RRRR (SEQ ID NO: 13); RRRRR (SEQ ID NO:14), KR, RK, KKR, KRK, RKK, KRR, RRK, (K/R) (K/R) (K/R) (K/R) (SEQ ID NO:15), (K/R) (K/R) (K/R) (K/R) (K/R) (SEQ ID NO:16), or any combination thereof. In some aspects, the ED comprises KK, KKK, KKKKKK (SEQ ID NO:11), KKKKKKKKK (SEQ ID NO:12), or any combination thereof. In some aspects, ND comprises an amino acid sequence as set forth in G: X2: X3: X4: X5: X6, wherein G represents Gly; wherein ": represents a peptide bond; wherein each of X2 to X6 independently represents an amino acid; wherein X6 represents a basic amino acid. In some aspects, the X6 amino acid is selected from the group consisting of: lys, Arg, and His. In some aspects, the X5 amino acid is selected from the group consisting of: pro, Gly, Ala and Ser. In some aspects, the X2 amino acid is selected from the group consisting of: pro, Gly, Ala and Ser. In some aspects, X4 is selected from the group consisting of: pro, Gly, Ala, Ser, Val, Ile, Leu, Phe, Trp, Tyr, Gln, and Met.

In some aspects, a scaffold moiety (e.g., scaffold Y) comprises an N-terminal domain (ND) and an Effector Domain (ED), wherein ND comprises an amino acid sequence as set forth in G: X2: X3: X4: X5: X6, wherein G represents Gly; wherein ": represents a peptide bond; wherein each of X2 through X6 is independently an amino acid; wherein X6 comprises a basic amino acid, and wherein ED is linked to X6 by a peptide bond and comprises at least one lysine at the N-terminus of ED.

In some aspects, ND of the scaffold moiety (e.g., scaffold Y) comprises the amino acid sequence G: X2: X3: X4: X5: X6, wherein G represents Gly; ": represents a peptide bond; x2 represents an amino acid selected from the group consisting of Pro, Gly, Ala and Ser; x3 represents any amino acid; x4 represents an amino acid selected from the group consisting of Pro, Gly, Ala, Ser, Val, Ile, Leu, Phe, Trp, Tyr, Gln, and Met; x5 represents an amino acid selected from the group consisting of Pro, Gly, Ala and Ser; x6 represents an amino acid selected from the group consisting of Lys, Arg and His.

In some aspects, the X3 amino acid is selected from the group consisting of: asn, Gln, Ser, Thr, Asp, Glu, Lys, His, and Arg.

In some aspects, ND and ED are connected by a linker. In some aspects, the linker comprises one or more amino acids. In some aspects, the term "linker" refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) or a non-polypeptide, such as an alkyl chain. In some aspects, two or more linkers can be connected in series. Generally, the joint provides flexibility or prevents/improves space obstruction. The joint is not typically cut; however, in certain aspects, such cutting may be desirable. Thus, in some aspects, the linker may comprise one or more protease cleavable sites, which may flank the linker within the linker sequence or at either end of the linker sequence. When ND and ED are linked by a linker, ED comprises at least two lysines, at least three lysines, at least four lysines, at least five lysines, at least six lysines or at least seven lysines. Useful linkers for connecting ND and ED are disclosed elsewhere in this specification.

In some aspects, a scaffold moiety (e.g., scaffold Y) comprises ND-ED, wherein: ND comprises G, X2, X3, X4, X5, X6; wherein: g represents Gly; ": represents a peptide bond; x2 represents an amino acid selected from the group consisting of Pro, Gly, Ala and Ser; x3 represents any amino acid; x4 represents an amino acid selected from the group consisting of Pro, Gly, Ala, Ser, Val, Ile, Leu, Phe, Trp, Tyr, Glu, and Met; x5 represents an amino acid selected from the group consisting of Pro, Gly, Ala and Ser; x6 represents an amino acid selected from the group consisting of Lys, Arg and His; "-" represents an optional linker; ED is an effector domain comprising (i) at least two consecutive lysines (Lys) linked by a peptide bond or one or more amino acids to X6, or (ii) at least one lysine linked directly by a peptide bond to X6.

In some aspects, X2 amino acid is selected from the group consisting of Gly and Ala. In some aspects, the X3 amino acid is Lys. In some aspects, the X4 amino acid is Leu or Glu. In some aspects, the X5 amino acid is selected from the group consisting of Ser and Ala. In some aspects, the X6 amino acid is Lys. In some aspects, X2 amino acid is Gly, Ala, or Ser; x3 amino acid is Lys or Glu; x4 amino acid is Leu, Phe, Ser, or Glu; x5 amino acid is Ser or Ala; the X6 amino acid is Lys. In some aspects, a "-" linker comprises a peptide bond or one or more amino acids.

In some aspects, the ED in the scaffold moiety comprises Lys (K), KK, KKK, KKKKKK (SEQ ID NO:11), KKKKKKKKK (SEQ ID NO:12), Arg (R), RR, RRR, RRRR (SEQ ID NO: 13); RRRRR (SEQ ID NO:14), KR, RK, KKR, KRK, RKK, KRR, RRK, (K/R) (K/R) (K/R) (K/R) (SEQ ID NO:15), (K/R) (K/R) (K/R) (K/R) (K/R) (SEQ ID NO:16), or any combination thereof.

In some aspects, the scaffold moiety (e.g., scaffold Y) comprises an amino acid sequence selected from the group consisting of: (i) GGKLSKK (SEQ ID NO:17), (ii) GAKLSKK (SEQ ID NO:18), (iii) GGKQSKK (SEQ ID NO:19), (iv) GGKLAKK (SEQ ID NO:20), or (v) any combination thereof.

In some aspects, ND in the scaffold moiety (e.g., scaffold Y) comprises an amino acid sequence selected from the group consisting of: (i) GGKLSK (SEQ ID NO:51), (ii) GAKLSK (SEQ ID NO:52), (iii) GGKQSK (SEQ ID NO:53), (iv) GGKLAK (SEQ ID NO:54), and (v) any combination thereof; and the ED in the scaffold protein comprises an amino acid sequence selected from the group consisting of: K. KK, KKK, KKKG (SEQ ID NO:55), KKKGY (SEQ ID NO:56), KKKGYN (SEQ ID NO:57), KKKGYNV (SEQ ID NO:58), KKKGYVN (SEQ ID NO:59), KKKGYS (SEQ ID NO:60), KKKGYG (SEQ ID NO:61), KKKGYGG (SEQ ID NO:62), KKKGKGS (SEQ ID NO:63), KKKGSG (SEQ ID NO:64), KKKGSGS (SEQ ID NO:66), KKKS (SEQ ID NO:67), KKKSG (SEQ ID NO:68), KKKSGG (SEQ ID NO:69), KKGGS (SEQ ID NO:70), KKGGSG (SEQ ID NO:71), KKSGGSGG (SEQ ID NO:72), KKKSGGSGGS (SEQ ID NO:73), KRFSKS (SEQ ID NO:241), and any combination thereof.

In some aspects, the polypeptide sequence useful for the scaffold Y of the present disclosure consists of an amino acid sequence selected from the group consisting of: (i) GGKLSKK (SEQ ID NO:21), (ii) GAKLSKK (SEQ ID NO:18), (iii) GGKQSKK (SEQ ID NO:19), (iv) GGKLAKK (SEQ ID NO:20), or (v) any combination thereof.

In some aspects, the scaffold moiety (e.g., scaffold Y) comprises an amino acid sequence selected from the group consisting of: (i) GGKLSKKK (SEQ ID NO:22), (ii) GGKLSKKS (SEQ ID NO:23), (iii) GAKLSKKK (SEQ ID NO:24), (iv) GAKLSKKS (SEQ ID NO:25), (v) GGKQSKKK (SEQ ID NO:26), (vi) GGKQSKKS (SEQ ID NO:27), (vii) GGKLAKKK (SEQ ID NO:28), (viii) GGKLAKKS (SEQ ID NO:29), and (ix) any combination thereof.

In some aspects, the polypeptide sequences useful for the scaffold moieties of the disclosure (e.g., scaffold Y) consist of an amino acid sequence selected from the group consisting of: (i) GGKLSKKK (SEQ ID NO:22), (ii) GGKLSKKS (SEQ ID NO:23), (iii) GAKLSKKK (SEQ ID NO:24), (iv) GAKLSKKS (SEQ ID NO:25), (v) GGKQSKKK (SEQ ID NO:26), (vi) GGKQSKKS (SEQ ID NO:27), (vii) GGKLAKKK (SEQ ID NO:28), (viii) GGKLAKKS (SEQ ID NO:29), and (ix) any combination thereof. In some aspects, the scaffold proteins of the present disclosure comprise at least two consecutive lysines (Lys) in sequence.

In some aspects, the length of a stent portion (e.g., stent Y) is at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, at least about 50, at least about 55, at least about 60, at least about 65, at least about 50, at least about 30, or about 30, at least about 30, or at least about 30, or a, At least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 105, at least about 110, at least about 115, at least about 120, at least about 125, at least about 130, at least about 135, at least about 140, at least about 145, at least about 150, at least about 155, at least about 160, at least about 165, at least about 170, at least about 175, at least about 180, at least about 185, at least about 190, at least about 195, at least about 200, at least about 205, at least about 210, at least about 215, at least about 220, at least about 225, at least about 230, at least about 235, at least about 240, at least about 245, at least about 250, at least about 255, at least about 260, at least about 265, at least about 270, at least about 275, at least about 280, at least about 285, at least about 290, at least about 295, at least about 300, at least about 305, at least about 310, at least about 315, at least about 320, at least about 325, at least about 300, at least about, At least about 330, at least about 335, at least about 340, at least about 345, or at least about 350 amino acids.

In some aspects, the scaffold moiety (e.g., scaffold Y) is between about 5 and about 10 amino acids, between about 10 and about 20 amino acids, between about 20 and about 30 amino acids, between about 30 and about 40 amino acids, between about 40 and about 50 amino acids, between about 50 and about 60 amino acids, between about 60 and about 70 amino acids, between about 70 and about 80 amino acids, between about 80 and about 90 amino acids, between about 90 and about 100 amino acids, between about 100 and about 110 amino acids, between about 110 and about 120 amino acids, between about 120 and about 130 amino acids, between about 130 and about 140 amino acids, between about 140 and about 150 amino acids, between about 150 and about 160 amino acids, between about 160 and about 170 amino acids, between about 170 and about 180 amino acids, between about 180 and about 190 amino acids, a scaffold Y is between, Between about 190 and about 200 amino acids, between about 200 and about 210 amino acids, between about 210 and about 220 amino acids, between about 220 and about 230 amino acids, between about 230 and about 240 amino acids, between about 240 and about 250 amino acids, between about 250 and about 260 amino acids, between about 260 and about 270 amino acids, between about 270 and about 280 amino acids, between about 280 and about 290 amino acids, between about 290 and about 300 amino acids, between about 300 and about 310 amino acids, between about 310 and about 320 amino acids, between about 320 and about 330 amino acids, between about 330 and about 340 amino acids, or between about 340 and about 250 amino acids.

In some aspects, the scaffold moiety (e.g., scaffold Y) comprises (i) GGKLSKKKKGYNVN (SEQ ID NO:32), (ii) GAKLSKKKKGYNVN (SEQ ID NO:33), (iii) GGKQSKKKKGYNVN (SEQ ID NO:34), (iv) GGKLAKKKKGYNVN (SEQ ID NO:35), (v) GGKLSKKKKGYSGG (SEQ ID NO:36), (vi) GGKLSKKKKGSGGS (SEQ ID NO:37), (vii) GGKLSKKKKSGGSG (SEQ ID NO:38), (viii) GGKLSKKKSGGSGG (SEQ ID NO:39), (ix) GGKLSKKSGGSGGS (SEQ ID NO:40), (x) GGKLSKSGGSGGSV (SEQ ID NO:41), or (xi) GAKKSKKRFSFKKS (SEQ ID NO: 42).

In some aspects, the polypeptide sequences for the scaffold moieties of the disclosure (e.g., scaffold Y) consist of (i) GGKLSKKKKGYNVN (SEQ ID NO:32), (ii) GAKLSKKKKGYNVN (SEQ ID NO:33), (iii) GGKQSKKKKGYNVN (SEQ ID NO:34), (iv) GGKLAKKKKGYNVN (SEQ ID NO:35), (v) GGKLSKKKKGYSGG (SEQ ID NO:36), (vi) GGKLSKKKKGSGGS (SEQ ID NO:37), (vii) GGKLSKKKKSGGSG (SEQ ID NO:38), (viii) GGKLSKKKSGGSGG (SEQ ID NO:39), (ix) GGKLSKKSGGSGGS (SEQ ID NO:40), (x) GGKLSKSGGSGGSV (SEQ ID NO:41), or (xi) GAKKSKKRFSFKKS (SEQ ID NO: 42).

Non-limiting examples of stent portions (e.g., stent Y) that can be used in the present disclosure are listed below. In some aspects, the scaffold moiety (e.g., scaffold Y) comprises the group of amino acid sequences listed in table 4. In some aspects, a scaffold moiety (e.g., scaffold Y) consists of the group of amino acid sequences listed in table 4.

TABLE 4 exemplary Stent portions

In some aspects, a scaffold moiety useful in the present disclosure (e.g., scaffold Y) does not comprise an N-terminal Met. In some aspects, the scaffold moiety (e.g., scaffold Y) comprises a lipidated amino acid, such as a myristoylated amino acid, at the N-terminus of the scaffold protein, which can be used as a lipid. In some aspects, the amino acid residue at the N-terminus of the scaffold protein is Gly. The presence of an N-terminal glycine is an absolute requirement for N-myristoylation. In some aspects, the amino acid residues at the N-terminus of the scaffold protein are synthetic. In some aspects, the amino acid residue at the N-terminus of the scaffold protein is a glycine analog, such as allylglycine, butylglycine, or propargylglycine.

In other aspects, the lipid can be any lipid known in the art, such as palmitic acid or glycosylphosphatidylinositol. In unusual cases, for example, by using media that is conditioned with myristic acid, some other fatty acids, including short chain and unsaturated fatty acids, can be attached to the N-terminal glycine. For example, in the BK channel, myristate is reported to be linked post-translationally to internal serine/threonine or tyrosine residues via hydroxyl ester bonds. The membrane fractions that can be used as scaffold fractions known in the art are listed in the table below.

Table 5: modifying group

G.3 scaffold protein fusion constructs

In some aspects, the scaffold moiety is linked to one or more heterologous proteins. One or more heterologous proteins may be attached to the N-terminus of the scaffold moiety. One or more heterologous proteins may be attached to the C-terminus of the scaffold moiety. In some aspects, one or more heterologous proteins are linked to the N-terminus and C-terminus of the scaffold moiety. In some aspects, the heterologous protein is a mammalian protein. In some aspects, the heterologous protein is a human protein.

In some aspects, a scaffold moiety may be used to attach any moiety to the luminal and/or outer surface of an exosome. For example, in addition to the outer surface of an EV (e.g., exosome), a PTGFRN polypeptide may be used to link biologically active molecules inside a cavity (e.g., on the surface of the cavity). Thus, in certain aspects, the scaffold moiety can serve a dual purpose, e.g., a bioactive molecule on the luminal surface of an EV (e.g., exosome) and a second bioactive molecule or other payload on the outer surface, or a bioactive molecule on the outer surface of an exosome and a second bioactive molecule or other payload on the luminal surface of an EV (e.g., exosome).

G.4 lipids

Suitable scaffold moieties capable of attaching a bioactive molecule to the surface of an EV (e.g., exosome) by chemical attachment to a maleimide moiety include, for example, sterols (e.g., cholesterol), phospholipids, lysophospholipids, fatty acids, or lipid-soluble vitamins, as detailed below.

In some aspects, the scaffold moiety can be a lipid. The lipid scaffold moiety may be any lipid known in the art, such as palmitic acid or glycosylphosphatidylinositol. In some aspects, the lipid is a fatty acid, a phospholipid (e.g., phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine), or an analog thereof (e.g., phosphatidylcholine, lecithin, phosphatidylethanolamine, cephalin, or phosphatidylserine, or an analog or portion thereof, such as a partially hydrolyzed portion thereof).

The scaffold moiety can be attached (e.g., chemically attached) to the bioactive molecule using a maleimide moiety. Such linkage may be directly or indirectly through a linker or a combination of linkers, at any chemically feasible location, such as at the 5 'and/or 3' end of a nucleotide sequence of, for example, a biologically active molecule (e.g., an ASO). In one aspect, the scaffold moiety is only attached (e.g., chemically attached via a maleimide moiety) to the 3' terminus of the biologically active molecule. In one aspect, the scaffold moiety is only attached (e.g., chemically attached via a maleimide moiety) to, for example, the 5' end of the nucleotide sequence of the biologically active molecule (e.g., ASO). In one aspect, the scaffold moiety is not attached (e.g., chemically attached via a maleimide moiety) at either a position such as the 3 'end or the 5' end of the nucleotide sequence of the biologically active molecule (e.g., ASO).

In some aspects, the bioactive molecule can be linked (e.g., chemically linked via a maleimide moiety), directly or indirectly linked via a linker, for example, to any lipid disclosed above (e.g., palmitic acid, myristic acid, fatty acid, farnesyl, geranyl-geranyl, or cholesterol). In some aspects, the scaffold moiety may comprise two or more types of scaffold moieties disclosed herein. For example, in some aspects, the scaffold moiety may comprise two lipids, e.g., one phospholipid and one fatty acid, or two phospholipids, or two fatty acids, or one lipid and one vitamin, or one cholesterol and one vitamin, and the like. They collectively have 6-80 carbon atoms (i.e., an Equivalent Carbon Number (ECN) of about 6 to about 80).

In some aspects, a combination of scaffold moieties, e.g., a lipid (e.g., fatty acid), has an ECN of about 6 to about 80, about 8 to about 80, about 10 to about 80, about 12 to about 80, about 14 to about 80, about 16 to about 80, about 18 to about 80, about 20 to about 80, about 22 to about 80, about 24 to about 80, about 26 to about 80, about 28 to about 80, about 30 to about 80, about 4 to about 76, about 6 to about 76, about 8 to about 76, about 10 to about 76, about 12 to about 76, about 14 to about 76, about 16 to about 76, about 18 to about 76, about 20 to about 76, about 22 to about 76, about 24 to about 76, about 26 to about 76, about 28 to about 76, about 30 to about 76, about 6 to about 72, about 8 to about 72, about 10 to about 72, about 12 to about 72, about 14 to about 72, about 16 to about 72, about 24 to about 72, about 24 to about 72, about 24 to about 72, about 24 to about 24, about 72, about 24 to about 76, about 24, about 76, about 24, about 6 to about 24, about 76, about 6 to about 76, about 6 to about 76, about 72, about 6 to about 72, about 6 to about 72, about 76, about 72, about 76, about 6 to about 72, about 6 to about 72, about 6 to about 76, about 72, about 76, about 72, about 6 to about 24, about 6 to about 72, about 6 to about 72, about 24, about 6 to about 72, about 80, about 6 to about 80, about 24, about 80, About 26 to about 72, about 28 to about 72, about 30 to about 72, about 6 to about 68, about 8 to about 68, about 10 to about 68, about 12 to about 68, about 14 to about 68, about 16 to about 68, 1 about 8 to about 68, about 20 to about 68, about 22 to about 68, about 24 to about 68, about 26 to about 68, about 28 to about 68, about 30 to about 68, about 6 to about 64, about 8 to about 64, about 10 to about 64, about 12 to about 64, about 14 to about 64, about 16 to about 64, about 18 to about 64, about 20 to about 64, about 22 to about 64, about 24 to about 64, about 26 to about 64, about 28 to about 64, about 30 to about 64, about 6 to about 60, about 8 to about 60, about 10 to about 60, about 12 to about 56, about 14 to about 56, about 16 to about 56, about 18 to about 56, about 22 to about 56, about 24 to about 24, about 24 to about 56, about 24 to about 52, about 24 to about 24, about 24 to about 56, about 24 to about 24, about 24 to about 52, about 24 to about 24, about 24 to about 24, about 24 to about 24, about 68, about 24 to about 24, about 64, about 24 to about 24, about 24, about 24 to about 68, about 24 about 64, about 24 about 64, about 24, about 64, about 24 about 64, about 24 about 60, about 24 about 60, About 8 to about 52, about 10 to about 52, about 12 to about 52, about 14 to about 52, about 16 to about 52, about 18 to about 52, about 20 to about 52, about 22 to about 52, about 24 to about 52, about 26 to about 52, about 28 to about 52, about 30 to about 52, about 6 to about 48, about 8 to about 48, about 10 to about 48, about 12 to about 48, about 14 to about 48, 1 about 6 to about 48, 1 about 8 to about 48, about 20 to about 48, 2 about 2 to about 48, about 24 to about 48, about 26 to about 48, about 28 to about 48, about 30 to about 48, about 6 to about 44, about 8 to about 44, about 10 to about 44, about 12 to about 44, about 14 to about 44, about 16 to about 44, about 18 to about 44, about 20 to about 44, about 22 to about 44, about 24 to about 44, about 6 to about 44, about 30 to about 40, about 40 to about 40, about 40 to about 48, about 6 to about 48, about 44, about 6 to about 44, about 6, about 44, about 6 to about 44, about 6 to about 44, about 6 to about 6, About 16 to about 40, about 18 to about 40, about 20 to about 40, 2 about 2 to about 40, about 24 to about 40, about 26 to about 40, 2 about 8 to about 40, about 30 to about 40, about 6 to about 36, about 8 to about 36, about 10 to about 36, about 12 to about 36, about 14 to about 36, about 16 to about 36, about 18 to about 36, about 20 to about 36, about 22 to about 36, about 24 to about 36, about 26 to about 36, about 28 to about 36, about 30 to about 36, about 6 to about 32, about 8 to about 32, 1 about 0 to about 32, about 12 to about 32, about 14 to about 32, 1 about 6 to about 32, 1 about 8 to about 32, about 20 to about 32, about 22 to about 32, about 24 to about 32, about 26 to about 32, 28 to about 32, or about 30 to about 32.

II.G.3.a Cholesterol and other sterols

In some aspects, the scaffold moiety comprises a sterol, a steroid, an hopane, a hydroxysteroid, a ring-opened steroid, or their lipophilic analogs. In some aspects, the scaffold moiety comprises a sterol, such as a plant sterol, a mold sterol, or an animal sterol. Exemplary zoosterols include cholesterol and 24S-hydroxycholesterol; exemplary phytosterols include ergosterol (mycopasterol), campesterol, sitosterol, and stigmasterol. In some aspects, the sterol is selected from the group consisting of ergosterol, 7-dehydrocholesterol, cholesterol, 24S-hydroxycholesterol, lanosterol, cycloartenol, fucosterol, sarinosterol, campesterol, beta-sitosterol, sitostanol, coprostanol, avenasterol or stigmasterol. Sterols can be present as free sterols, acylated (sterol esters), alkylated (sterol alkyl ethers), sulfated (sterol sulfates), or linked to a glycoside moiety (sterol glycosides), which itself can be acylated (acylated sterol glycosides).

In some aspects, the scaffold moiety comprises a steroid. In some aspects, the steroid is selected from dihydrotestosterone, ursanol, agave sapogenin, dioscin, progesterone, or cortisol.

For example, the sterol can be conjugated to the bioactive molecule directly or through a combination of linkers at available-OH groups of the sterol. Exemplary sterols have the general skeleton shown below:

as another example, ergosterol has the following structure:

cholesterol has the following structure:

thus, in some aspects, the free-OH group of the sterol or steroid is used to conjugate the bioactive molecule (e.g., ASO) to the sterol (e.g., cholesterol) or steroid, either directly or through a combination of linkers.

II.G.3.b fatty acids

In some aspects, the scaffold moiety comprises a fatty acid. In some aspects, the fatty acid is a short, medium, or long chain fatty acid. In some aspects, the fatty acid is a saturated fatty acid. In some aspects, the fatty acid is an unsaturated fatty acid. In some aspects, the fatty acid is a monounsaturated fatty acid. In some aspects, the fatty acid is a polyunsaturated fatty acid, such as an omega-3 (omega-3) or omega-6 (omega-6) fatty acid.

In some aspects, the lipid (e.g., fatty acid) has a C₂-C₆₀And (3) a chain. In some aspects, the lipid (e.g., fatty acid) has a C₂-C₂₈And (3) a chain. In some aspects, the fatty acid has C₂-C₄₀And (3) a chain. In some aspects, the fatty acid has C₂-C₁₂Or C₄-C₁₂And (3) a chain. In some aspects, the fatty acid has C ₄-C₄₀And (3) a chain. In some aspects, the fatty acid has C₄-C₄₀、C₂-C₃₈、C₂-C₃₆、C₂-C₃₄、C₂-C₃₂、C₂-C₃₀、C₄-C₃₀、C₂-C₂₈、C₄-C₂₈、C₂-C₂₆、C₄-C₂₆、C₂-C₂₄、C₄-C₂₄、C₆-C₂₄、C₈-C₂₄、C₁₀-C₂₄、C₂-C₂₂、C₄-C₂₂、C₆-C₂₂、C₈-C₂₂、C₁₀-C₂₂、C₂-C₂₀、C₄-C₂₀、C₆-C₂₀、C₈-C₂₀、C₁₀-C₂₀、C₂-C₁₈、C₄-C₁₈、C₆-C₁₈、C₈-C₁₈、C₁₀-C₁₈、C₁₂-C₁₈、C₁₄-C₁₈、C₁₆-C₁₈、C₂-C₁₆、C₄-C₁₆、C₆-C₁₆、C₈-C₁₆、C₁₀-C₁₆、C₁₂-C₁₆、C₁₄-C₁₆、C₂-C₁₅、C₄-C₁₅、C₆-C₁₅、C₈-C₁₅、C₉-C₁₅、C₁₀-C₁₅、C₁₁-C₁₅、C₁₂-C₁₅、C₁₃-C₁₅、C₂-C₁₄、C₄-C₁₄、C₆-C₁₄、C₈-C₁₄、C₉-C₁₄、C₁₀-C₁₄、C₁₁-C₁₄、C₁₂-C₁₄、C₂-C₁₃、C₄-C₁₃、C₆-C₁₃、C₇-C₁₃、C₈-C₁₃、C₉-C₁₃、C₁₀-C₁₃、C₁₀-C₁₃、C₁₁-C₁₃、C₂-C₁₂、C₄-C₁₂、C₆-C₁₂、C₇-C₁₂、C₈-C₁₂、C₉-C₁₂、C₁₀-C₁₂、C₂-C₁₁、C₄-C₁₁、C₆-C₁₁、C₇-C₁₁、C₈-C₁₁、C₉-C₁₁、C₂-C₁₀、C₄-C₁₀、C₂-C₉、C₄-C₉、C₂-C₈、C₂-C₇、C₄-C₇、C₂-C₆Or C₄-C₆And (3) a chain. In some aspects, the fatty acid has C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、C₂₀、C₂₁、C₂₂、C₂₃、C₂₄、C₂₅、C₂₆、C₂₇、C₂₈、C₂₉、C₃₀、C₃₁、C₃₂、C₃₃、C₃₄、C₃₅、C₃₆、C₃₇、C₃₈、C₃₉、C₄₀、C₄₁、C₄₂、C₄₃、C₄₄、C₄₅、C₄₆、C₄₇、C₄₈、C₄₉、C₅₀、C₅₁、C₅₂、C₅₃、C₅₄、C₅₅、C₅₆、C₅₇、C₅₈、C₅₉Or C₆₀And (3) a chain.

In some aspects, the scaffold moiety comprises two fatty acids, wherein each is independently selected from fatty acids having a chain comprising any of the foregoing ranges or numbers of carbon atoms. In some aspects, one of the fatty acids is independently a fatty acid having a C6-C21 chain, and one of the fatty acids is independently a fatty acid having a C12-C36 chain. In some aspects, each fatty acid independently has a chain of about 11, about 12, about 13, about 14, about 15, about 16, about 17, or about 18 carbon atoms.

Suitable fatty acids include saturated straight chain fatty acids, saturated branched chain fatty acids, unsaturated fatty acids, hydroxy fatty acids, and polycarboxylic acids. In some aspects, such fatty acids have up to about 32 carbon atoms.

Examples of useful saturated straight chain fatty acids include those having an even number of carbon atoms, such as butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, lignoceric, hexacosanoic, octacosanoic, triacontanoic, and triacontanoic acids, and those having an odd number of carbon atoms, such as propionic, n-valeric, heptanoic, pelargonic, undecanoic, tridecanoic, pentadecanoic, heptadecanoic, nonadecanoic, heneicosanoic, pentacosanoic, and heptacosanoic acids.

Examples of suitable saturated branched fatty acids include isobutyric acid, isocaproic acid, isooctanoic acid, isodecanoic acid, isolauric acid, 11-methyldodecanoic acid, isomyristic acid, 13-methyl-tetradecanoic acid, isopalmitic acid, 15-methyl-hexadecanoic acid, isostearic acid, 17-methyloctadecanoic acid, isoarachidic acid, 19-methyl-eicosanoic acid, alpha-ethyl-hexanoic acid, alpha-hexyldecanoic acid, alpha-heptylundecanoic acid, 2-decyltetradecanoic acid, 2-undecyltetradecanoic acid, 2-decyltecanoic acid, 2-undecylpentadecanoic acid, and Fine oxocol 1800acid (a product of Nissan Chemical Industries, Ltd.). Suitable saturated odd-carbon branched fatty acids include anteiso fatty acids terminated with isobutyl groups, such as 6-methyl-octanoic acid, 8-methyl-decanoic acid, 10-methyl-dodecanoic acid, 12-methyl-tetradecanoic acid, 14-methyl-hexadecanoic acid, 16-methyl-octadecanoic acid, 18-methyl-eicosanoic acid, 20-methyl-docosanoic acid, 22-methyl-tetracosanoic acid, 24-methyl-hexacosanoic acid and 26-methyl-octacosanoic acid.

Examples of suitable unsaturated fatty acids include 4-decenoic acid, hexanoic acid, 4-dodecenoic acid, 5-dodecenoic acid, lauroleic acid, 4-tetradecenoic acid, 5-tetradecenoic acid, 9-tetradecenoic acid, palmitoleic acid, 6-octadecenoic acid, oleic acid, 9-octadecenoic acid, 11-octadecenoic acid, 9-eicosenoic acid, cis-11-eicosenoic acid, cetoleic acid, 13-docosenoic acid, 15-tetracosenoic acid, 17-hexacosenoic acid, 6,9,12, 15-hexadecatetraenoic acid, linoleic acid, linolenic acid, alpha-eleostearic acid, beta-eleostearic acid, punicic acid, 6,9,12, 15-octadecatetraenoic acid, stearidonic acid, 5,8,11, 14-eicosatetraenoic acid, 5,8,11,14, 17-eicosapentaenoic acid, 7,10,13,16, 19-docosapentaenoic acid, 4,7,10,13,16, 19-docosahexaenoic acid, etc.

Examples of suitable hydroxy fatty acids include alpha-hydroxy lauric acid, alpha-hydroxy myristic acid, alpha-hydroxy palmitic acid, alpha-hydroxy stearic acid, omega-hydroxy lauric acid, alpha-hydroxy arachidic acid, 9-hydroxy-12-octadecenoic acid, ricinoleic acid, alpha-hydroxydocosanoic acid, 9-hydroxy-trans-10, 12-octadecadienoic acid, glycyrrhizic acid, isopropanoic acid, 9, 10-dihydroxystearic acid, 12-hydroxystearic acid, and the like.

Examples of suitable polycarboxylic acids include oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, D, L-malic acid, and the like.

In some aspects, each fatty acid is independently selected from propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nonadecanoic acid, arachidic acid, behenic acid, tricosanic acid, lignoceric acid, pentacosanoic acid, cerotic acid, heptacosanoic acid, montanic acid, nonacosanoic acid, melissic acid, hendecanoic acid, costumic acid, triacontanoic acid, cerotic acid, triacontanoic acid, or triacontanoic acid.

In some aspects, each fatty acid is independently selected from alpha-linolenic acid, stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, gamma-linoleic acid, dihomo-gamma-linoleic acid, arachidonic acid, docosatetraenoic acid, palmitoleic acid, vaccenic acid, eleostearic acid, oleic acid, elaidic acid, eicosenoic acid, erucic acid, nervonic acid, midic acid, adrenic acid, primary pentaenoic acid, capone acid, sardine acid, herring acid, docosahexaenoic acid, or tetracosapentaenoic acid, or other mono-or polyunsaturated fatty acids.

In some aspects, one or both fatty acids are essential fatty acids. In view of the beneficial health effects of certain essential fatty acids, the therapeutic benefit of the disclosed therapeutic-loaded exosomes can be increased by including such fatty acids in the therapeutic. In some aspects, the essential fatty acid is an n-6 or n-3 essential fatty acid selected from the group consisting of: linolenic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, adrenic acid, docosapentaen n-6 acid, alpha-linolenic acid, stearic acid, 20:4n-3 acid, eicosapentaenoic acid, docosapentaen n-3 acid or docosahexaenoic acid.

In some aspects, each fatty acid is independently selected from the group consisting of all-cis-7, 10, 13-hexadecatrienoic acid, alpha-linolenic acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid (EPA), docosapentaenoic acid, docosahexaenoic acid (DHA), tetracosapentaenoic acid, tetracosahexaenoic acid, or lipoic acid. In other aspects, the fatty acid is selected from eicosapentaenoic acid, docosahexaenoic acid, or lipoic acid. Other examples of fatty acids include all-cis-7, 10, 13-hexadecatrienoic acid, alpha-linolenic acid (ALA or all-cis-9, 12, 15-octadecatrienoic acid), stearic acid (STD or all-cis-6, 9,12, 15-octadecatetraenoic acid), eicosatrienoic acid (ETE or all-cis-11, 14, 17-eicosatrienoic acid), arachidonic acid (ETA or all-cis-8, 11,14, 17-eicosatetraenoic acid), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA, docosapentaenoic acid or all-cis-7, 10,13,16, 19-docosapentaenoic acid), docosahexaenoic acid (DHA or all-cis-4, 7,10,13,16, 19-docosahexaenoic acid), eicosapentaenoic acid (all-cis-9, 12,15,18, 21-docosahexaenoic acid) or docosahexaenoic acid (azelaic acid or holo-cis-6, 9,12,15,18, 21-tetracosenoic acid). In some aspects, the fatty acid is a medium chain fatty acid, such as lipoic acid.

The chain lengths of fatty acid chains vary widely and can be classified according to chain length, for example from short to long. Short Chain Fatty Acids (SCFA) are fatty acids having about five or fewer carbon chains (e.g., butyric acid). In some aspects, the fatty acid is SCFA. Medium Chain Fatty Acids (MCFA), including fatty acids having about 6-12 carbon chains, can form medium chain triglycerides. In some aspects, the fatty acid is MCFA. Long Chain Fatty Acids (LCFA) include fatty acids having a carbon chain of 13-21. In some aspects, the fatty acid is LCFA. In some aspects, the fatty acid is LCFA. Very Long Chain Fatty Acids (VLCFA) include fatty acids having 22 or more carbon chains, such as between about 22 and about 60, between about 22 and about 50, or between about 22 and about 40. In some aspects, the fatty acid is VLCFA.

II.G.3.c Phospholipids

In some aspects, the scaffold moiety comprises a phospholipid. Phospholipids are a class of lipids that are the major components of all cell membranes. Due to their amphiphilic character, they can form lipid bilayers. The structure of phospholipid molecules generally consists of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group. For example, the phospholipid may be a lipid according to the formula:

Wherein R is_pRepresents a phospholipid moiety, R₁And R₂Represents fatty acid moieties with or without the same or different unsaturation.

The phospholipid moiety may be selected from, for example, the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.

Specific phospholipids may promote fusion with lipid bilayers, e.g., the lipid bilayer of exosome membranes. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of the membrane. The fusion of the phospholipid to the membrane may allow one or more components of the lipid-containing composition to bind to or cross the membrane.

The fatty acid moiety may be selected from the non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

The phospholipids used as scaffold moieties in the present disclosure may be natural or non-natural phospholipids. Non-natural phospholipid species are also contemplated, including natural species with modifications and substitutions, including branching, oxidation, cyclization, and alkyne. For example, the phospholipid may be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are substituted with triple bonds). Under appropriate reaction conditions, the alkynyl group can undergo a copper-catalyzed cycloaddition reaction upon exposure to the azide.

Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol and phosphatidic acid.

Examples of phospholipids that can be used in the scaffold moieties disclosed herein include

Phosphatidylethanolamine: for example, dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, 1-palmitoyl-2-oleoyl phosphatidylethanolamine, 1-oleoyl-2-palmitoyl phosphatidylethanolamine, and diocanoyl phosphatidylethanolamine;

phosphatidylglycerol: for example, dilauroyl phosphatidyl glycerol, dimyristoyl phosphatidyl glycerol, dipalmitoyl phosphatidyl glycerol, distearoyl phosphatidyl glycerol, dioleoyl phosphatidyl glycerol, 1-palmitoyl-2-oleoyl-phosphatidyl glycerol, 1-oleoyl-2-palmitoyl-phosphatidyl glycerol, and dierucoyl phosphatidyl glycerol;

phosphatidylserine: for example, dilauroyl phosphatidylserine, dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, distearoyl phosphatidylserine, dioleoyl phosphatidylserine, 1-palmitoyl-2-oleoyl-phosphatidylserine, 1-oleoyl-2-palmitoyl-phosphatidylserine, and dioleoyl phosphatidylserine;

Phosphatidic acid: for example, dilauroylphosphatidic acid, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, distearoylphosphatidic acid, dioleoylphosphatidic acid, 1-palmitoyl-2-oleoylphosphatidic acid, 1-oleoyl-2-palmitoylphosphatidic acid, and dierucoylphosphatidic acid; and

phosphatidylinositol: for example, dilauroyl phosphatidylinositol, dimyristoyl phosphatidylinositol, dipalmitoyl phosphatidylinositol, distearoyl phosphatidylinositol, dioleoyl phosphatidylinositol, 1-palmitoyl-2-oleoyl-phosphatidylinositol, 1-oleoyl-2-palmitoyl phosphatidylinositol, and erucic acid phosphatidylinositol.

The phospholipids may be of the symmetric or asymmetric type. As used herein, the term "symmetric phospholipid" includes glycerophospholipids and sphingolipids having matching fatty acid moieties, wherein the hydrocarbon chains of the variable fatty acid moieties and the sphingosine backbone include a significant number of carbon atoms. As used herein, the term "asymmetric phospholipid" includes lysolipidic, glycerophospholipids and sphingolipids having different fatty acid moieties (e.g., fatty acid moieties having different numbers of carbon atoms and/or degrees of unsaturation (e.g., double bonds)), wherein the hydrocarbon chains of the variable fatty acid moieties and the sphingosine backbone include different numbers of carbon atoms (e.g., the variable fatty acid moieties include at least two more carbon atoms than the hydrocarbon chains or at least two more carbon atoms than the hydrocarbon chains).

In some aspects, the scaffold moiety comprises at least one symmetric phospholipid. The symmetric phospholipids may be selected from the non-limiting group consisting of:

1, 2-dipropionyl-sn-glycero-3-phosphocholine (03:0 PC),

1, 2-dibutyryl-sn-glycero-3-phosphocholine (04:0 PC),

1, 2-dipentanyl-sn-glycero-3-phosphocholine (05:0 PC),

1, 2-dihexanoyl-sn-glycero-3-phosphocholine (06:0 PC),

1, 2-diheptanoyl-sn-glycero-3-phosphocholine (07:0 PC),

1, 2-dioctanoyl-sn-glycero-3-phosphocholine (08:0 PC),

1, 2-dinonylsn-glycero-3-phosphocholine (09:0 PC),

1, 2-didecanoyl-sn-glycero-3-phosphocholine (10:0 PC),

1, 2-di-undecanoyl-sn-glycero-3-phosphocholine (11:0 PC, DUPC),

1, 2-dilauroyl-sn-glycero-3-phosphocholine (12:0 PC),

1, 2-didecanoyl-sn-glycero-3-phosphocholine (13:0 PC),

1, 2-dimyristoyl-sn-glycero-3-phosphocholine (14:0 PC, DMPC),

1, 2-dipentadecacarbonyl-sn-glycero-3-phosphocholine (15:0 PC),

1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (16:0 PC, DPPC),

1, 2-diphytanoyl-sn-glycero-3-phosphocholine (4ME 16:0 PC),

1, 2-di-heptadecacarbonyl-sn-glycero-3-phosphocholine (17:0 PC),

1, 2-distearoyl-sn-glycerol-3-phosphocholine (18:0 PC, DSPC),

1, 2-di-nonadecanoyl-sn-glycero-3-phosphocholine (19:0 PC),

1, 2-di-arachidonyl-sn-glycero-3-phosphocholine (20:0 PC),

1, 2-dieicosancarbonyl-sn-glycero-3-phosphocholine (21:0 PC),

1, 2-di-docosanoyl-sn-glycero-3-phosphocholine (22:0 PC),

1, 2-di-tricosancarbonyl-sn-glycero-3-phosphocholine (23:0 PC),

1, 2-di-pyrolusioyl-sn-glycero-3-phosphocholine (24:0 PC),

1, 2-dimyristoyl-sn-glycero-3-phosphocholine (14:1 (. DELTA.9-Cis) PC),

1, 2-dimyristoyl-sn-glycero-3-phosphocholine (14:1 (. DELTA.9-Trans) PC),

1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (16:1 (. DELTA.9-Cis) PC),

1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (16:1 (. DELTA.9-Trans) PC),

1, 2-dilinoleoyl-sn-glycero-3-phosphocholine (18:1 (. DELTA.6-Cis) PC),

1, 2-di-Cis-oleoyl-sn-glycero-3-phosphocholine (18:1 (. DELTA.9-Cis) PC, DOPC),

1, 2-dioleoyl-sn-glycero-3-phosphocholine (18:1 (. DELTA.9-Trans) PC),

1, 2-dioleoyl-sn-glycero-3-phosphocholine (18:2(Cis) PC, DLPC),

1, 2-dioleyl-sn-glycero-3-phosphocholine (18:3(Cis) PC, DLnPC),

1, 2-di-eicosanoyl-sn-glycero-3-phosphocholine (20:1(Cis) PC),

1, 2-di-arachidonyl-sn-glycero-3-phosphocholine (20:4(Cis) PC, DAPC),

1, 2-dioleoyl-sn-glycero-3-phosphocholine (22:1(Cis) PC),

1, 2-docosahexaenoyl-sn-glycero-3-phosphocholine (22:6(Cis) PC, DHAPC),

1, 2-diacyl-sn-glycero-3-phosphocholine (24:1(Cis) PC),

1, 2-dihexanoyl-sn-glycero-3-phosphoethanolamine (06:0PE),

1, 2-dioctanoyl-sn-glycero-3-phosphoethanolamine (08: 0PE),

1, 2-didecanoyl-sn-glycero-3-phosphoethanolamine (10: 0PE),

1, 2-dilauroyl-sn-glycero-3-phosphoethanolamine (12: 0PE),

1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (14: 0PE),

1, 2-dipentadecacarbonyl-sn-glycero-3-phosphoethanolamine (15: 0PE),

1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (16: 0PE),

1, 2-diphytoyl-sn-glycero-3-phosphoethanolamine (4ME 16: 0PE),

1, 2-di-heptadecacarbonyl-sn-glycero-3-phosphoethanolamine (17: 0PE),

1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine (18: 0PE, DSPE),

1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (16:1 PE),

1, 2-di-Cis-oleoyl-sn-glycero-3-phosphoethanolamine (18:1 (. DELTA.9-Cis) PE, DOPE),

1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (18:1 (. DELTA.9-Trans) PE),

1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (18:2 PE, DLPE),

1, 2-dioleyl-sn-glycero-3-phosphoethanolamine (18:3 PE, DLnPE),

1, 2-dianhydrotetraallyl-sn-glycero-3-phosphoethanolamine (20:4 PE, DAPE),

1, 2-docosahexenoyl-sn-glycero-3-phosphoethanolamine (22:6 PE, DHAPE),

1, 2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC),

1, 2-dioleoyl-sn-glycerol-3-phospho-rac- (1-glycerol) sodium salt (DOPG) and any combination thereof.

In some aspects, the scaffold moiety comprises at least one symmetric phospholipid selected from the non-limiting group consisting of: DLPC, DMPC, DOPC, DPPC, DSPC, DUPC, 18:0 diether PC, DLnPC, DAPC, DHAPC, DOPE, 4ME 16:0 PE, DSPE, DLPE, DLnPE, DAPE, DHAPE, DOPG, and any combination thereof.

In some aspects, the scaffold moiety comprises at least one asymmetric phospholipid. The asymmetric phospholipid may be selected from the non-limiting group consisting of:

1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (14:0-16:0 PC, MPPC),

1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (14:0-18:0 PC, MSPC),

1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine (16:0-02:0 PC),

1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (16:0-14:0 PC, PMPC),

1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (16:0-18:0 PC, PSPC),

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0-18:1 PC, POPC),

1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2 PC, PLPC),

1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (16:0-20:4 PC),

1-palmitoyl-2-docosahexenoyl-sn-glycero-3-phosphocholine (14:0-22:6 PC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (18:0-14:0 PC, SMPC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:0-16:0 PC, SPPC),

1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0-18:1 PC, SOPC),

1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0-18:2 PC),

1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (18:0-20:4 PC),

1-stearoyl-2-docosahexenoyl-sn-glycerol-3-phosphocholine (18:0-22:6 PC),

1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine (18:1-14:0 PC, OMPC),

1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:1-16:0 PC, OPPC),

1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (18:1-18:0 PC, OSPC),

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1 PE, POPE),

1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:2 PE),

1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (16:0-20:4 PE),

1-palmitoyl-2-docosahexenoyl-sn-glycero-3-phosphoethanolamine (16:0-22:6 PE),

1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:1 PE),

1-stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:2 PE),

1-stearoyl-2-arachidonyl-sn-glycero-3-phosphoethanolamine (18:0-20:4 PE),

1-stearoyl-2-docosahexaenoyl-sn-glycerol-3-phosphoethanolamine (18:0-22:6 PE),

1-oleoyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (ochemsPC) and

any combination thereof.

To provide more significant nuclease resistance, cellular uptake efficiency, and more significant RNA interference effects, phosphatidylethanolamine can be used as a scaffold moiety, such as dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, and dioleoyl phosphatidylethanolamine.

The binding site of the lipid (e.g., phospholipid) and the linker or the bioactive molecule (e.g., ASO) may be appropriately selected according to the types of the lipid and the linker or the bioactive molecule. Any position other than the hydrophobic group of the lipid may be linked to the linker or the bioactive molecule by a chemical bond. For example, when phosphatidylethanolamine is used, the linkage may be formed by forming an amide bond or the like between the amino group of phosphatidylethanolamine and a linker or a bioactive molecule.

When phosphatidylglycerol is used, the linkage may be formed by forming an ester bond, an ether bond, or the like between the hydroxyl group of the glycerol residue and the linker or the bioactive molecule.

When phosphatidylserine is used, the linkage may be formed by forming an amide bond, an ester bond, or the like between the amino group or carboxyl group of the serine residue and the linker or the biologically active molecule.

When phosphatidic acid is used, the linkage may be formed by forming a phosphoester bond or the like between the phosphate residue and the linker or the biologically active molecule.

When phosphatidylinositol is used, the linkage may be formed by forming an ester bond, an ether bond, or the like between the hydroxyl group of the inositol residue and the linker or the bioactive molecule.

II.G.3.d lyso-lipid (e.g. lyso-phospholipid)

In some aspects, the scaffold moiety comprises a lysolipid, such as a lysophospholipid. Lysolipids are derivatives of lipids in which one or both fatty acyl chains are typically removed by hydrolysis. Lysophospholipids are derivatives of phospholipids in which one or both fatty acyl chains are removed by hydrolysis.

In some aspects, the scaffold moiety comprises any of the phospholipids disclosed above, wherein one or both acyl chains are removed by hydrolysis, and the resulting lysophospholipid comprises one or no fatty acid acyl chain.

In some aspects, the scaffold moiety comprises a lysoglycerophospholipid, a lysoglycosphingolipid, a lysophosphatidylcholine, a lysophosphatidylethanolamine, a lysophosphatidylinositol, or a lysophosphatidylserine.

In some aspects, the scaffold moiety comprises a lysolipid selected from the non-limiting group consisting of:

1-hexanoyl-2-hydroxy-sn-glycero-3-phosphocholine (06:0 Lyso PC),

1-heptanoyl-2-hydroxy-sn-glycero-3-phosphocholine (07:0 Lyso PC),

1-octanoyl-2-hydroxy-sn-glycero-3-phosphocholine (08:0 Lyso PC),

1-nonanoyl-2-hydroxy-sn-glycero-3-phosphocholine (09:0 Lyso PC),

1-decanoyl-2-hydroxy-sn-glycero-3-phosphocholine (10:0 Lyso PC),

1-undecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (11:0 Lyso PC),

1-lauroyl-2-hydroxy-sn-glycero-3-phosphocholine (12:0 Lyso PC),

1-tridecyl-2-hydroxy-sn-glycero-3-phosphocholine (13:0 Lyso PC),

1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (14:0 Lyso PC),

1-pentadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (15:0 Lyso PC),

1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (16:0 Lyso PC),

1-heptadecacarbonyl-2-hydroxy-sn-glycero-3-phosphocholine (17:0 Lyso PC),

1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (18:0 Lyso PC),

1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (18:1 Lyso PC),

1-nonadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (19:0 Lyso PC),

1-eicosanyl-2-hydroxy-sn-glycero-3-phosphocholine (20:0 Lyso PC),

1-docosanyl-2-hydroxy-sn-glycero-3-phosphocholine (22:0 Lyso PC),

1-tetracosanyl-2-hydroxy-sn-glycero-3-phosphocholine (24:0 Lyso PC),

1-hexacosanyl-2-hydroxy-sn-glycero-3-phosphocholine (26:0 Lyso PC),

1-myristoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (14:0 Lyso PE),

1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (16:0 Lyso PE),

1-stearoyl-2-hydroxy-sn-glycerol-3-phosphoethanolamine (18:0 Lyso PE),

1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (18:1 Lyso PE),

1-hexadecyl-sn-glycerol-3-phosphocholine (C16 Lyso PC) and

any combination thereof.

II.G.3.e vitamins

In some aspects, the scaffold moiety comprises a lipophilic vitamin, such as folic acid, vitamin a, vitamin E, or vitamin K.

In some aspects, the scaffold portion comprises vitamin a. Vitamin a is a group of unsaturated nutritional organic compounds including retinol, retinal, retinoic acid and several provitamin a carotenoids (most notably β -carotene). In some aspects, the scaffold moiety comprises retinol. In some aspects, the scaffold moiety comprises a retinoid. Retinoids are a class of compounds that act as, or are chemically related to, isovitamins of vitamin a. In some aspects, the scaffold moiety comprises a first generation retinoid (e.g., retinol, tretinoin, isotretinoin, or alitretinoin), a second generation retinoid (e.g., an retinol ester or avilamgic acid), a third generation retinoid (e.g., adapalene, bexarotene, or tazarotene), or any combination thereof.

First generation retinoids

Retinol

Second generation retinoids

Etriptylate

Acitretin

Third generation retinoids

Adapalene

Bexarotene

Tazarotene

In some aspects, the scaffold portion comprises vitamin E. Tocopherols are a class of methylated phenols, many of which have vitamin E activity. Thus, in some aspects, the scaffold moiety comprises alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, or a combination thereof.

Alpha tocopherol

Beta tocopherol

Gamma tocopherol

Delta tocopherol

Tocotrienols also have vitamin E activity. The key chemical structural difference between tocotrienols and tocopherols is that tocotrienols have an unsaturated isoprenoid side chain with three carbon-carbon double bonds, whereas the side chain of tocopherols is saturated. In some aspects, the scaffold moiety comprises alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, delta-tocotrienol, or a combination thereof. The tocotrienol can be represented by the following formula

alpha (α) -tocotrienol: r1 ═ Me, R2 ═ Me, R3 ═ Me;

beta (β) -tocotrienol: r1 ═ Me, R2 ═ H, R3 ═ Me;

gamma (γ) -tocotrienol: r1 ═ H, R2 ═ Me, R3 ═ Me;

delta (δ) -tocotrienol: r1 ═ H, R2 ═ H, and R3 ═ Me.

In some aspects, the scaffold moiety comprises vitamin K. Chemically, the vitamin K family comprises 2-methyl-1.4-naphthoquinone (3-) derivatives. Vitamin K includes two natural vitamins: vitamin K₁And vitamin K₂. Vitamin K₁(also known as phytoketodione, phylloquinone or (E) -phytoketodione) is characterized by the presence of a phytyl group. Vitamin K₂The structure of (menaquinone) is indicated by the presence of a polyisoprenyl side chain in the molecule, which may contain from six to 13 prenyl units. Thus, vitamin K₂Consisting of a number of related chemical subtypes, with carbon side chains of varying lengths consisting of isoprenoid atom groups. MK-4 is the most common vitamin K₂Form (a). Long chain forms, such as MK-7, MK-8 and MK-9, predominate in fermented foods. Vitamin K in longer chain form₂(such as MK-10 through MK-13) are synthesized by bacteria, but they are poorly absorbed and have little biological function. In addition to the natural form of vitamin K, there are various synthetic forms of vitamin K, such as vitamin K₃(menadione; 2-methylnaphthalene-1, 4-dione), vitamin K₄And vitamin K₅。

Thus, in some aspects, the scaffold moiety comprises vitamin K ₁、K₂(e.g., MK-4, MK-5, MK-6, MK-7, MK-8, MK-9, MK-10, MK-11, MK-12 or MK-13), K₃、K₄、K₅Or any combination thereof.

G.5 chemically induced dimers

In some aspects, a scaffold moiety (e.g., a scaffold protein) is linked to a binding partner of a chemically-induced dimer. In certain aspects, a scaffold moiety (e.g., a scaffold protein) is linked to a binding partner of a chemically-induced dimer, and a bioactive molecule is linked to the corresponding binding partner. In these aspects, the scaffold moiety (e.g., scaffold protein) and the biologically active molecule bind to each other in the presence of a chemical that induces dimerization of the binding partners. In some aspects, the binding partner is attached to the N-terminus of the scaffold moiety. In some aspects, the binding partner is attached to the C-terminus of the scaffold moiety (e.g., scaffold protein). In some aspects, the binding partner is attached to a luminal domain of the scaffold moiety (e.g., scaffold protein).

In certain aspects, a scaffold moiety (e.g., a scaffold protein) is linked to an affinity agent. In some aspects, the affinity agent is attached to the N-terminus of the scaffold moiety (e.g., scaffold protein). In some aspects, the affinity agent is attached to the C-terminus of the scaffold moiety (e.g., a scaffold protein). In some aspects, the affinity agent is attached to a cavity domain of a scaffold moiety (e.g., a scaffold protein). In some aspects, the affinity agent comprises a polypeptide capable of binding to a biologically active molecule. In some aspects, the affinity agent comprises a receptor. In some aspects, the affinity agent comprises an antibody or antigen binding domain, as disclosed herein. In some aspects, the affinity agent binds to one or more bioactive molecules.

In some aspects, the interaction between the affinity agent and the bioactive molecule is transient. In some aspects, the bioactive molecule dissociates from the affinity agent under certain conditions. In certain aspects, the affinity of an affinity agent for a bioactive molecule is pH dependent. In some aspects, the bioactive molecule dissociates from the affinity agent at a pH of at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, or at least about 12. In some aspects, the affinity of the affinity agent for the bioactive molecule is dependent on the concentration of calcium, magnesium, sulfate, phosphate, or any combination thereof in a solution comprising the bioactive molecule and the affinity agent. In some aspects, the affinity of an affinity agent for a bioactive molecule is dependent on the salt concentration and/or ionic strength of the solution comprising the bioactive molecule and the affinity agent. In some aspects, the bioactive molecule and the affinity agent are dissociable under reducing conditions.

In some aspects, a scaffold moiety (e.g., a scaffold protein) is linked to a polypeptide that can bind to a biologically active molecule. In some aspects, the binding polypeptide is attached to the N-terminus of the scaffold moiety (e.g., scaffold protein). In some aspects, the binding polypeptide is attached to the C-terminus of a scaffold moiety (e.g., a scaffold protein). In some aspects, the binding polypeptide is linked to a cavity domain of a scaffold moiety (e.g., a scaffold protein)

In some aspects, the binding polypeptide comprises an antigen binding domain. In some aspects, the antigen binding domain comprises an antigen binding fragment of an antibody. In some aspects, the antigen binding domain comprises a single chain antibody or antigen binding fragment thereof. In some aspects, the antigen binding domain comprises a humanized antibody or antigen binding fragment thereof. In some aspects, the antigen binding domain comprises a murine antibody or antigen binding fragment thereof. In some aspects, the antigen binding domain comprises a chimeric antibody (e.g., a mouse-human, mouse-primate, or primate-human monoclonal antibody) or an antigen binding fragment thereof. In some aspects, the antigen binding domain comprises a camelid antibody, a shark IgNAR, or an antigen binding fragment of an anti-idiotypic antibody. In some aspects, the antigen binding domain comprises a camelid antibody or an antigen binding fragment thereof. In some aspects, the antigen binding domain comprises shark IgNAR or an antigen binding fragment thereof. In some aspects, the antigen binding domain comprises an anti-idiotype antibody or antigen binding fragment thereof.

In some aspects, the antigen binding domain comprises a single chain antibody. In some aspects, the antigen binding domain comprises a scFv. In some aspects, the antigen binding domain comprises (scFv) ₂. In some aspects, the antigen binding domain comprises a Fab. In some aspects, the antigen binding domain comprises a Fab'. In some aspects, the antigen binding domain comprises F (ab')₂. In some aspects, the antigen binding domain comprises F (ab1)₂. In some aspects, the antigen binding domain comprises an Fv. In some aspects, the antigen binding domain comprises a dAb. In some aspects, the antigen binding domain comprises a single chain Fab. In some aspects, the antigen binding domain comprises an Fd fragment.

In some aspects, the antigen binding domain comprises a dimer. In some aspects, the antigen binding domain comprises a minibody. In some aspects, the antigen binding domain comprises an antibody-associated polypeptide. In a particular aspect, the antigen binding domain comprises a nanobody.

In some aspects, a scaffold moiety (e.g., a scaffold protein) is linked to an Fc receptor, and a bioactive molecule is linked to an Fc. In certain aspects, the Fc receptor is an Fc γ receptor selected from Fc γ receptor I (Fc γ R1), Fc γ RIIA, Fc γ IIB, Fc γ IIIA, and Fc γ IIIB; fc is the Fc of IgG. In certain aspects, the Fc receptor is Fc γ R1, and the Fc is that of IgG. In some aspects, the Fc receptor is Fc α receptor I (Fc α R1), and wherein Fc is Fc for IgA. In some aspects, the Fc receptor is an fcepsilon receptor selected from fcepsilon receptor I (fcepsilon RI) and fcepsilon RII, and wherein Fc is Fc for IgE.

In some aspects, a scaffold moiety (e.g., a scaffold protein) is attached to a nanobody; and the biologically active molecule is linked to an immunoglobulin constant region (Fc). In certain aspects, the nanobody specifically binds to Fc.

Preparation method

The EVs (e.g., exosomes) of the present disclosure may be produced by chemical synthesis, recombinant DNA techniques, biochemical or enzymatic fragmentation of larger molecules, a combination of the above, or by any other method. In one aspect, the present disclosure provides a method of conjugating a biologically active molecule to an EV (e.g., exosome). The method comprises attaching a biologically active molecule to an EV (e.g., exosome) via a maleimide moiety as described above.

In addition to amine-reactive compounds, those compounds having chemical groups that form bonds with sulfhydryl groups (-SH) are the most common crosslinkers and modifying reagents for proteins and other bioconjugation techniques. Sulfhydryl groups, also known as thiols, are present in the side chains of cysteine (Cys, C) amino acids in proteins. Cysteine sulfhydryl pairs are typically linked by disulfide bonds (-S-S-) within or between polypeptide chains as the basis of the native tertiary or quaternary protein structure. Generally, only free or reduced sulfhydryl groups (-SH) [ rather than sulfur atoms in disulfide bonds ] are available for reaction with thiol-reactive compounds.

Sulfhydryl groups are useful targets for protein conjugation and labeling. First, sulfhydryl groups are present in most proteins, but not as much as primary amines; thus, crosslinking by thiol groups is more selective and precise. Second, sulfhydryl groups in proteins are often associated with disulfide bonds, so that cross-linking of these sites does not usually significantly alter the underlying protein structure or block binding sites. Third, the amount of available (i.e., free) sulfhydryl groups can be readily controlled or modified; they can be generated by reduction of the native disulfide bond or can be introduced into the molecule by reaction with a primary amine using a thiol addition reagent such as 2-iminothiol (Traut's reagent), SATA, SATP or SAT (PEG). Finally, combining thiol-reactive groups with amine-reactive groups to prepare heterobifunctional crosslinkers provides greater flexibility and control over the crosslinking procedure. For example, using NHS ester 3-maleimidopropionate, which contains a maleimide group and a NHS ester, the NHS ester can be used to label primary amines (-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The maleimide group will react with the thiol group to form a covalent bond, linking the biomolecule to the thiol bond.

When the pH value of the reaction mixture is between 6.5 and 7.5, the maleimide group and the sulfhydryl group are specifically reacted; the result is the formation of an irreversible stable thioether bond (i.e., the bond cannot be cleaved with a reducing agent). Under more basic conditions (pH >8.5), the reaction favors primary amines and also increases the rate of hydrolysis of the maleimide groups to non-reactive maleamic acid. Maleimide does not react with tyrosine, histidine or methionine.

Thiol-containing compounds such as Dithiothreitol (DTT) and β -mercaptoethanol (BME) must be excluded from the reaction buffer used with maleimide because they compete for the conjugation site. For example, if DTT is used to reduce disulfide bonds in proteins to make thiol groups available for conjugation, the DTT must be completely removed using a desalting column before the maleimide reaction is initiated. Interestingly, the disulfide reducing agent TCEP is thiol-free and does not have to be removed prior to the reaction involving the maleimide reagent.

The excess maleimide can be quenched at the end of the reaction by addition of free thiol. EDTA may be included in the conjugation buffer to sequester stray divalent metals that would otherwise promote oxidation of sulfhydryl groups (non-reactivity).

In one aspect, linking includes treating the EV (e.g., exosomes) with a reducing agent. Suitable reducing agents include, for example, TCEP (tris (2-carboxyethyl) phosphine), DTT (dithiothreitol), BME (2-mercaptoethanol), thiolating agents, and any combination thereof. The thiolating agent may include, for example, Traut's reagent (2-iminothiol).

Following treatment with the reducing agent, the ligation reaction further comprises contacting the reduced EV (e.g., exosomes) with a maleimide moiety. In one aspect, the maleimide moiety is attached to the bioactive molecule prior to attachment to the EV (e.g., exosome). In some aspects, the maleimide moiety is further attached to a linker to attach the maleimide moiety to the biologically active molecule. Thus, in some aspects, one or more linkers or spacers are interposed between the maleimide moiety and the biologically active molecule.

Therapeutic use

The present disclosure provides methods of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a composition comprising an EV (e.g., exosome) of the present disclosure. The present disclosure also provides methods of preventing or ameliorating a symptom of a disease or disorder in a subject in need thereof, the method comprising administering to the subject a composition comprising an EV (e.g., exosome) of the present disclosure. Also provided are methods of diagnosing a disease or disorder in a subject in need thereof, the methods comprising administering to the subject a composition comprising an EV (e.g., exosome) of the present disclosure.

In one aspect, the disease or disorder is cancer, an inflammatory disease, a neurodegenerative disease, a central nervous disease, or a metabolic disease.

The present disclosure also provides methods of preventing and/or treating a disease or disorder in a subject in need thereof, the methods comprising administering an EV (e.g., exosome) disclosed herein to the subject. In some aspects, the disease or disorder that can be treated with the present methods includes cancer, graft versus host disease (GvHD), autoimmune disease, infectious disease, or fibrotic disease. In some aspects, the treatment is prophylactic. In other aspects, the EVs (e.g., exosomes) used in the present disclosure are used to induce an immune response. In other aspects, the EVs (e.g., exosomes) for use in the present disclosure are used to vaccinate a subject.

In some aspects, the disease or disorder is cancer. When administered to a subject with cancer, in certain aspects, EVs (e.g., exosomes) of the present disclosure may up-regulate the immune response and enhance tumor targeting of the subject's immune system. In some aspects, the cancer treated is characterized by infiltration of leukocytes (T cells, B cells, macrophages, dendritic cells, monocytes) into the tumor microenvironment or so-called "hot tumors" or "inflammatory tumors". In some aspects, the cancer treated is characterized by infiltration of low or undetectable levels of leukocytes into the tumor microenvironment, or so-called "cold tumors" or "non-inflammatory tumors". In some aspects, the EV (e.g., exosomes) is administered in an amount and for a duration sufficient to convert a "cold tumor" to a "hot tumor," i.e., the administration results in infiltration of leukocytes (such as T cells) into the tumor microenvironment. In certain aspects, the cancer comprises bladder cancer, cervical cancer, renal cell carcinoma, testicular cancer, colorectal cancer, lung cancer, head and neck cancer, as well as ovarian cancer, lymphoma, liver cancer, glioblastoma, melanoma, myeloma, leukemia, pancreatic cancer, or a combination thereof. In other aspects, the term "distal tumor" or "distant tumor" refers to a tumor that spreads from the original (or primary) tumor to distant organs or tissues, such as lymph nodes. In some aspects, the EVs (e.g., exosomes) of the present disclosure treat tumors after metastatic spread.

In some aspects, the disease or disorder is graft versus host disease (GvHD). In some aspects, the disease or disorder that can be treated with the present disclosure is an autoimmune disease. Non-limiting examples of autoimmune diseases include: multiple sclerosis, peripheral neuritis, sjogren's syndrome, rheumatoid arthritis, alopecia, autoimmune pancreatitis, behcet's disease, bullous pemphigoid, celiac disease, devycke's disease (neuromyelitis optica), glomerulonephritis, IgA nephropathy, vasculitis, scleroderma, diabetes, arteritis, vitiligo, ulcerative colitis, irritable bowel syndrome, psoriasis, uveitis, systemic lupus erythematosus, and combinations thereof.

In some aspects, the disease or disorder is an infectious disease. In certain aspects, the disease or disorder is an oncogenic virus. In some aspects, infectious diseases that can be treated with the present disclosure include, but are not limited to, human gammaherpes virus 4 (epstein-barr virus), influenza a virus, influenza b virus, cytomegalovirus, staphylococcus aureus, mycobacterium tuberculosis, chlamydia trachomatis, HIV-1, HIV-2, coronaviruses (e.g., MERS-CoV and SARS CoV), filoviruses (e.g., marburg virus and ebola virus), streptococcus pyogenes, streptococcus pneumoniae, plasmodium (e.g., plasmodium vivax and plasmodium falciparum), boroncus, Human Papilloma Virus (HPV), hepatitis b, hepatitis c, human herpes virus 8, herpes simplex virus 2(HSV2), klebsiella, pseudomonas aeruginosa, enterococcus, proteobacterium, enterobacter, actinomyces, coagulase-negative staphylococcus (costs), Mycoplasma or combinations thereof.

In some aspects, the EV (e.g., exosome) is administered intravenously to the circulatory system of the subject. In some aspects, the EV (e.g., exosomes) are infused into a suitable liquid and administered into a vein of the subject.

In some aspects, the EV (e.g., exosome) is administered intra-arterially to the circulatory system of the subject. In some aspects, an EV (e.g., exosome) is infused into a suitable liquid and administered into an artery of a subject.

In some aspects, the EV (e.g., exosome) is administered to the subject by intrathecal administration. In some aspects, the EV (e.g., exosomes) are administered by injection into the spinal canal or subarachnoid space so that it reaches the cerebrospinal fluid (CSF).

In some aspects, the EV (e.g., exosome) is administered intratumorally into one or more tumors of the subject.

In some aspects, the EV (e.g., exosomes) are administered to the subject by intranasal administration. In some aspects, the EV (e.g., exosomes) may be insufflated through the nose in a form for local administration or systemic administration. In certain aspects, the EV (e.g., exosomes) are administered as a nasal spray.

In some aspects, the EV (e.g., exosomes) are administered to the subject by intraperitoneal administration. In some aspects, the EV (e.g., exosomes) are infused into a suitable liquid and injected into the subject's peritoneum. In some aspects, intraperitoneal administration results in EV (e.g., exosomes) distribution to the lymph. In some aspects, intraperitoneal administration results in EV (e.g., exosomes) distribution to the thymus, spleen, and/or bone marrow. In some aspects, intraperitoneal administration results in the distribution of EVs (e.g., exosomes) to one or more lymph nodes. In some aspects, intraperitoneal administration results in EV (e.g., exosome) distribution to one or more of the cervical, inguinal, mediastinal, or sternal lymph nodes. In some aspects, intraperitoneal administration results in EV (e.g., exosomes) distribution to the pancreas.

In some aspects, the EV (e.g., exosome) is administered to the subject by periocular administration. In some aspects, the EV (e.g., exosomes) are injected into periocular tissue. Periocular drug administration includes subconjunctival, anterior sub-tenon's capsule, posterior sub-tenon's capsule, and retrobulbar routes of administration.

In some aspects, the EV (e.g., exosome) is administered intraocularly. Accordingly, the present disclosure provides a method of treating an ocular disease or disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising an Extracellular Vesicle (EV) of the present disclosure (e.g., an exosome), the composition comprising a payload (e.g., an AVV), wherein the administration of the composition is intraocular administration.

In some aspects, intraocular administration is selected from the group consisting of: intravitreal administration, intracameral administration, subconjunctival administration, subretinal administration, subscleral administration, intrachoroidal administration, and any combination thereof. In some aspects, intraocular administration includes injection of an EV (e.g., exosome) of the present disclosure. In some aspects, intraocular administration is intravitreal injection.

Pharmaceutical compositions and methods of administration

The present disclosure also provides pharmaceutical compositions comprising EVs (e.g., exosomes) described herein suitable for administration to a subject. The pharmaceutical compositions generally comprise a plurality of EVs (e.g., exosomes) comprising a biologically active molecule covalently attached to the plurality of EVs (e.g., exosomes) via a maleimide moiety and a pharmaceutically acceptable excipient or carrier in a form suitable for administration to a subject. The pharmaceutically acceptable excipient or carrier is determined, in part, by the particular composition being administered and the particular method used to administer the composition. Thus, there are a wide variety of suitable pharmaceutical composition formulations that comprise multiple EVs (e.g., exosomes). (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 18 th edition (1990)).

Pharmaceutical compositions are generally formulated aseptically and fully in accordance with all Good Manufacturing Practice (GMP) regulations of the U.S. food and drug administration. In some aspects, the pharmaceutical composition comprises one or more compounds, e.g., a small molecule covalently attached to an EV (e.g., an exosome), as described herein.

In some aspects, the pharmaceutical composition comprises one or more therapeutic agents and an EV (e.g., exosome), as described herein. In certain aspects, the EV (e.g., exosomes) are co-administered with one or more other therapeutic agents in a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition comprising an EV (e.g., exosome) is administered prior to administration of the additional therapeutic agent. In other aspects, the pharmaceutical composition comprising an EV (e.g., exosome) is administered after the administration of the additional therapeutic agent. In other aspects, the pharmaceutical composition comprising an EV (e.g., exosome) is administered concurrently with an additional therapeutic agent.

Provided herein are pharmaceutical compositions comprising an EV (e.g., exosome) of the present disclosure having a desired purity, and a pharmaceutically acceptable carrier or excipient in a form suitable for administration to a subject. The pharmaceutically acceptable excipient or carrier moiety may be determined by the particular composition being administered and by the particular method used to administer the composition. Thus, there is a wide variety of suitable formulations of pharmaceutical compositions comprising a plurality of extracellular vesicles. (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 21 st edition (2005)). Pharmaceutical compositions are generally formulated aseptically and fully in accordance with all Good Manufacturing Practice (GMP) regulations of the U.S. food and drug administration.

Acceptable carriers, excipients, or stabilizers are non-toxic to recipients (e.g., animals or humans) at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants (including ascorbic acid and methionine)Amino acids); preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides (e.g., sucrose or trehalose), and other carbohydrates (including glucose, mannose, or dextrins); chelating agents such as EDTA; sugar alcohols (e.g., mannitol or sorbitol); salt-forming counterions such as sodium ions; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEEN ^TM、PLURONICS^TMOr polyethylene glycol (PEG).

Examples of carriers or diluents include, but are not limited to, water, saline, ringer's solution, dextrose solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the extracellular vesicles described herein, its use in the compositions is contemplated. Supplemental therapeutic agents may also be incorporated into the composition. Typically, the pharmaceutical composition is formulated to be compatible with its intended route of administration. The EVs (e.g., exosomes) of the present disclosure may be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, intratumoral, intramuscular routes, or as inhalants. In certain aspects, a pharmaceutical composition comprising an EV (e.g., exosome) is administered intravenously (e.g., by injection). An EV (e.g., exosome) may optionally be administered in combination with other therapeutic agents that are at least partially effective in treating a disease, disorder, or condition to be treated by the EV (e.g., exosome).

The solution or suspension may comprise the following components: sterile diluents such as water, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and compounds for adjusting tonicity, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. The formulations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL^TM(BASF, Parsippany, n.j.) or Phosphate Buffered Saline (PBS). The compositions are generally sterile and fluid to the extent that easy injection is achieved. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like). Isotonic compounds, for example, sugars, polyols (such as mannitol, sorbitol), and sodium chloride may be added to the composition if desired. Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound that delays absorption (e.g., aluminum monostearate and gelatin).

Sterile injectable solutions can be prepared by mixing an effective amount of an EV (e.g., exosome) of the present disclosure in a suitable solvent with one or a combination of the components enumerated herein, as needed. Generally, dispersions are prepared by incorporating an EV (e.g., exosome) into a sterile vehicle containing a base dispersion medium and any required other components. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. EV (e.g., exosomes) may be administered in the form of a depot injection or implant formulation, which may be formulated in a manner that allows sustained or pulsed release of the EV (e.g., exosomes).

Systemic administration of EV (e.g., exosome) -containing compositions of the present disclosure may also be performed by transmucosal means. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of, for example, nasal sprays.

In certain aspects, a pharmaceutical composition comprising an EV (e.g., exosome) of the present disclosure is administered intravenously to a subject that would benefit from the pharmaceutical composition. In certain other aspects, the compositions are administered to the lymphatic system, for example, by intralymphatic injection or by intranodal injection (see, e.g., Senti et al, PNAS105(46):17908(2008)), or by intramuscular injection, by subcutaneous administration, by intratumoral injection, by direct injection into the thymus or into the liver.

In certain aspects, a pharmaceutical composition comprising an EV (e.g., exosome) of the present disclosure is administered as a liquid suspension. In certain aspects, the pharmaceutical composition is administered in the form of a formulation capable of forming a depot after administration. In certain preferred aspects, the depot slowly releases EV (e.g., exosomes), enters the circulation, or remains in depot form.

Typically, the pharmaceutically acceptable composition is highly purified to be contaminant-free, biocompatible and non-toxic, and suitable for administration to a subject. If water is a component of the vehicle, the water is highly purified and treated to be free of contaminants such as endotoxins.

The pharmaceutically acceptable carrier may be lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and/or mineral oil, but is not limited thereto. The pharmaceutical composition may further comprise lubricants, wetting agents, sweeteners, flavor enhancers, emulsifiers, suspending agents and/or preservatives.

The pharmaceutical compositions described herein comprise an EV (e.g., exosome) described herein and optionally a pharmaceutically active or therapeutic agent. The therapeutic agent may be a biologic agent, a small molecule agent, or a nucleic acid agent.

Dosage forms are provided that include a pharmaceutical composition comprising an EV (e.g., exosome) as described herein. In some aspects, the dosage form is formulated as a liquid suspension for intravenous injection. In some aspects, the dosage form is formulated as a liquid suspension for intratumoral injection.

In certain aspects, preparations of EVs (e.g., exosomes) of the present disclosure are subjected to radiation, such as X-rays, gamma rays, beta particles, alpha particles, neutrons, protons, elemental nuclei, ultraviolet light, to destroy residual replication-competent nucleic acids.

In certain aspects, a preparation of an EV (e.g., exosome) of the present disclosure is subjected to gamma radiation using a radiation dose of greater than about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 50, about 60, about 70, about 80, about 90, about 100, or greater than 100 kGy.

In certain aspects, preparations of EVs (e.g., exosomes) of the present disclosure are subjected to X-ray radiation using a radiation dose greater than about 0.1, about 0.5, about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, or about 10000.

The EVs (e.g., exosomes) of the present disclosure may be used concurrently with other drugs. In particular, the EVs (e.g., exosomes) of the present invention may be used with drugs such as hormonal therapeutic agents, chemotherapeutic agents, immunotherapeutic agents, drugs that inhibit the action of a cell growth factor or cell growth factor receptor, and the like.

VI. kit

The present disclosure also provides kits or articles of manufacture comprising one or more EVs (e.g., exosomes) of the present disclosure and optionally instructions for use. In some aspects, a kit or article of manufacture comprises a pharmaceutical composition described herein comprising at least one EV (e.g., exosome) of the present disclosure and instructions for use. In some aspects, a kit or article of manufacture comprises at least one EV (e.g., exosome) or pharmaceutical composition comprising an EV (e.g., exosome) of the present disclosure in one or more containers. One of skill in the art will readily recognize that the EVs of the present disclosure (e.g., exosomes), pharmaceutical compositions comprising the EVs of the present disclosure (e.g., exosomes), or combinations thereof, can be readily incorporated into one of the established kit formats, which are well known in the art.

In some aspects, a kit or article of manufacture comprises an EV (e.g., an exosome), one or more bioactive molecules, a reagent to covalently attach the one or more bioactive molecules to the EV (e.g., an exosome) via a maleimide moiety, or any combination thereof, and instructions to react to covalently attach the one or more bioactive molecules to the EV (e.g., an exosome) via a maleimide moiety.

In some aspects, the kit comprises reagents for conjugating a biologically active molecule to an EV (e.g., exosome) via a maleimide moiety, and instructions for performing the conjugation.

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. These techniques are explained fully in the literature. See, e.g., code of Sambrook et al, (1989) Molecular Cloning A Laboratory Manual (2 nd edition; Cold Spring Harbor Laboratory Press); compiled by Sambrook et al, (1992) Molecular Cloning, A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); glover eds, (1985) DNA Cloning, Volumes I and II; gait eds. (1984) Oligonucleotide Synthesis; mullis et al, U.S. patent nos. 4,683,195; coded by Hames and Higgins, (1984) Nucleic Acid Hybridization; coded by Hames And Higgins, (1984) transformation And transformation; freshney (1987) Culture Of Animal Cells (Alan r. loss, Inc.); immobilized Cells And Enzymes (IRL Press) (1986); perbal (1984) A Practical Guide To Molecular Cloning; the threading, Methods In Enzymology (Academic Press, Inc., N.Y.); miller and Calos eds (1987) Gene Transfer Vectors For Mammarian Cells, (Cold Spring Harbor Laboratory); wu et al, Methods In Enzymology, volumes 154 and 155; mayer And Walker, eds (1987) biochemical Methods In Cell And Molecular Biology (Academic Press, London); edited by Weir and Blackwell, (1986) Handbook Of Experimental Immunology, volumes I-IV; manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1986); crooke, Antisense drug Technology: Principles, Strategies and Applications, CRC Press version 2 (2007) and Ausubel et al (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

All references cited above, and all references cited herein, are hereby incorporated by reference in their entirety.

The following examples are provided by way of illustration and not by way of limitation.

Examples

The following examples are for illustrative purposes only and should not be construed as limiting the scope or content of the present invention in any way. The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E.Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); green and Sambrook et al, Molecular Cloning: A Laboratory Manual, 4 th edition (Cold Spring Harbor Laboratory Press, 2012); colowick and Kaplan, Methods In Enzymology (Academic Press); remington The Science and Practice of Pharmacy, 22 nd edition (Pharmaceutical Press, 2012); sundberg and Carey, Advanced Organic Chemistry: parts A and B, 5 th edition (Springer, 2007).

Example 1

Exosome isolation and loading

Exosome isolation: exosomes were collected from supernatants of HEK293SF cell high density suspension cultures after 7-9 days. Cell culture medium was centrifuged continuously, the supernatant of the previous centrifugation was used as input for the subsequent centrifugation: the cell culture medium was centrifuged at 5,000 Xg for 30 minutes, the supernatant was collected and the pellet was discarded; the supernatant was then centrifuged at 16,000 Xg for 30 minutes, the supernatant was collected and the pellet was discarded; the supernatant was then centrifuged at 133,900 Xg for 3 hours, the supernatant was discarded, and the pellet was collected and resuspended in 1mL PBS. Resuspended 133,900 Xg particles were passed through OPTIPREP ^TMFurther purification was run in iodixanol gradient: 4 layers sterile gradient by passing 3mL OPTIPREP^TM(60% iodixanol) was prepared by mixing with 1mL of resuspended pellet to yield 4mL of 45% iodixanol, then serially overlaying 3mL of 30% iodixanol, 2mL of 22.5% iodixanol, 2mL of 17.5% iodixanol, and 1mL of PBS in a 12mL Ultra-Clear (344059) tube for SW 41Ti rotors. The gradient was ultracentrifuged at 150,000 Xg for 16 hours at 4 ℃. Ultracentrifugation produces a top fraction known to contain exosomes, a middle fraction containing medium-density cell debris, and a bottom fraction containing high-density aggregates and cell debris. The exosome layer was then gently collected from about 2mL from the top of the tube.

The exosome fraction was diluted in about 32mL PBS in 38.5mL Ultra-Clear (344058) tubes and centrifuged at 10,000 × g for 30 min, the supernatant was collected and ultracentrifuged at 133,900 × g for 3 h at 4 ℃ to pellet the purified exosomes. The precipitated exosomes were then suspended in a minimum volume of PBS (about 200 μ Ι _) and stored at 4 ℃. The final purified concentration of exosomes was determined using nanoparticle chase analysis (NTA).

Exosome loading: to load exosomes with maleimide conjugates, exosomes were chemically reduced with TCEP (tris (2-carboxyethyl) phosphine hydrochloride) at a concentration of 1 to 50 mM; in some cases, the reducing step comprises or is preceded by treatment with 1-2M guanidine hydrochloride for one hour at room temperature. Exosomes were exchanged into PBS by dilution to 1mL in PBS, centrifugation at 100,000 × g for 20 min (TLA 120.2 rotor, Beckman) to pellet exosomes, supernatant was removed and discarded, and the pellet was resuspended in 1mL PBS; repeat once to ensure complete buffer exchange. The final exosome pellet was resuspended in 0.1mL PBS, to which the compound to be loaded was added up to a final concentration of 300 μ M. Exosomes were incubated overnight at 4 ℃ and then washed with PBS to remove compounds not conjugated to exosomes (diluted to 1mL in PBS, centrifuged at 100,000 × g for 20 min (TLA 120.2 rotor, Beckman) to pellet exosomes, supernatant removed and discarded, and pellet resuspended in 1mL PBS; repeated once to ensure complete buffer exchange).

Example 2

Efficacy of free and exosome-linked STING agonists

Figure 1B provides STING agonist compounds tested in PBMC assays. The compound was synthesized in Sygnature. PBMCs were isolated from heparinized human blood using standard protocols with Ficoll-Hypaque density gradients. For each condition to be tested, 500,000 PBMCs were seeded in one well of a 96-well plate and incubated overnight with the test samples. The next day, cells were centrifuged in the plate (500 × g, 10 min) and the supernatant was collected. The release of interferon beta (IFN β) into the cell culture supernatant was measured using ELISA. Fig. 2.

Figure 2 shows STING agonism of thiol or amine reactive compounds assessed by PBMC assay. PBMCs from three different healthy human donors were used to determine the activity of free compounds (filled circles) or compounds loaded on exosomes (open circles). All compounds with maleimide-linked chemistry (CP227, CP229, CP250) showed a 3-4 log increase in potency when associated with exosomes. Exosome binding by passive loading (CP232) showed an approximately 2-fold log increase in potency. Notably, succinimide ligation resulted in low loading and no induction of IFN β release was detected in the exosome-loaded sample (CP 246).

FIGS. 3A-3C show a comparison of thiol reactivity and lipid association chemistry of loading of STING agonists. PBMCs from two different healthy human donors were used to determine the activity of three compounds: CP227, CP229, and CP 238. These compounds were tested as free (blue circles/lines) or loaded on exosomes (green circles/lines). Both compounds containing thiol-reactive maleimide linker chemistry (CP227 and CP229) showed more than a 3-fold log increase in potency when attached to exosomes. In contrast, compounds containing lipid-bound cholesterol chemicals show less than a 1-fold log change in potency when conjugated to exosomes. Thus, maleimide attachment is superior to cholesterol.

FIGS. 4A-4C show a comparison of unmodified and thiol-reactive chemistries loaded with STING agonists. Exosomes passively loaded with cyclic dinucleotide STING agonists (ADUS100 and CL656) were compared to agonists chemically linked to exosomes with maleimide chemicals (the data for the maleimide compounds are the same as in figure 2; i.e., the compounds tested were CP227, CP229 and CP 238). EC of two load types₅₀The values are compared in the table shown in fig. 4. Typically, the exosome-mediated potency of maleimide-conjugated compounds is increased by more than 10-fold compared to unmodified compounds. Therefore, attachment of maleimide to exosomes is preferred to passive loading.

Figures 5A and 5B show a comparison of loading and activity of different STING agonists. The amount of STING agonist loaded onto exosomes using different attachment chemistries was quantified by mass spectrometry. STING agonist EC₅₀Values were calculated according to the human PBMC assay. The results indicate that STING agonist loading/activity varies from method to method.

Example 3

Efficacy of exosome-linked MMAE

FIG. 6 shows the structures of monomethyl auristatin E (MMAE) and maleimide-vc-PABC-MMAE (vc-MMAE). These two compounds were used to test the loading of cytotoxic compounds (MMAE) on exosomes. Both compounds are commercially available and ordered from MedChemExpress.

MMAE cytotoxicity was evaluated on RAW264.7(RAW) cells. See fig. 7. RAW cells are human macrophage lineage. Each well of a 96-well plate was seeded with 10,000 RAW cells and cell growth was monitored using the IncuCyte instrument, with the cells being imaged over a period of approximately 5 days. Free MMAE or DMSO (vehicle control) was added to RAW cells over a range of concentrations, and effective growth inhibition and cell death was observed when MMAE concentration was greater than 1.1 nM. The DMSO control showed no effect on cell viability or growth. Images show cells after 5 days of treatment with a prescribed dose of MMAE or DMSO; growth inhibition was evident from the reduction in cell numbers after treatment with MMAE, whereas cell death was indicated by the loose state of the cells (visible in the 10, 100 and 300nM images). MMAE has a steep dose-response curve.

The difference in MMAE potency in the presence or absence of the maleimide-Val-Cit-PABC linker is shown in figure 8A. RAW cells were treated with unmodified MMAE or vc-MMAE at the indicated concentrations. When administered as free drug to cells, vc-MMAE appeared to be about 100-fold less toxic than unmodified MMAE. Thus, the attachment of MMAE to the vc linker results in reduced efficacy.

Figure 8B shows exosome clearance after incubation with MMAE. Exosomes were incubated with MMAE overnight at 4 ℃ and then washed twice by ultracentrifugation (pellet at 100,000 × g for 20 min, resuspended in PBS, then pellet a second time, then resuspended). In the absence of testing, exosomes treated with MMAE were found to induce any growth inhibition or toxicity when added to RAW cells. This indicates that free MMAE does not bind significantly to exosomes and that cleanup procedures remove MMAE from exosomes.

Figure 8C shows that the vc-MMAE loaded exosomes exhibit potent biological activity. Exosomes were chemically reduced with TCEP (5mM) and guanidine hydrochloride (Gdn) at concentrations ranging from 0.1 to 2M. Exosomes were cleared by ultracentrifugation (pelleted at 100,000 × g for 20 min, resuspended in PBS, then pelleted a second time, then resuspended) and then added to RAW cells at the indicated MOI (number of exosomes per cell). Toxicity was assessed by measuring inhibition of cell growth after 5 days of culture. RAW cells showed a dose-dependent decrease in proliferation after treatment with vc-MMAE loaded exosomes. Thus, vc-MMAE was significantly attached to exosomes and was not washed by chaotropic agents and centrifugation. Furthermore, MMAE linked to exosomes showed potent inhibition of cell growth.

Figure 8D shows that vc-MMAE activity requires chemical reduction of exosomes. Exosomes were either kept in PBS or treated with PBS containing 5mM TCEP, then incubated with MMAE or vc-MMAE. The samples were cleared by ultracentrifugation and then added to RAW cells. Cell growth was significantly inhibited only by TCEP-reduced exosomes incubated with vc-MMAE (dark red triangles). This indicates that the maleimide group on the vc-MMAE compound is conjugated to the thiol group generated on the exosome after chemical reduction.

Figure 9A shows the effect of reducing conditions and loading concentrations of compounds on exosome potency. Exosomes were treated with a series of reducing conditions (0-50mM TCEP with or without 1M guanidine hydrochloride), cleaned by ultracentrifugation, and incubated overnight at 4 ℃ with 10 or 100 μ M vc-MMAE. Exosomes were cleared by ultracentrifugation and then added to RAW cells to test their effect on cell growth. Exosomes that were not chemically reduced by TCEP treatment had no effect on cell growth. In comparison under reducing conditions, loading with 100 μ M vc-MMAE resulted in a significant increase in potency compared to loading with 10 μ M vc-MMAE. In the presence of 1M Gdn, all concentrations of TCEP produced similar potency, except for the highest concentration of TCEP (50mM), which showed reduced potency. Increasing the concentration of TCEP from 1.5mM to 15mM in the absence of guanidine hydrochloride resulted in increased potency; 50mM TCEP also showed reduced efficacy in the absence of guanidine hydrochloride.

Similar to fig. 9A, fig. 9B shows the effect of reducing conditions and loading concentrations of the compound on exosome potency, but at higher vc-MMAE concentrations. The same experimental conditions as for the experiment shown in fig. 9A were used, but 100 and 300 μ M vc-MMAE were used as loading concentrations. Similar to what was observed with 10 and 100 μ M vc-MMAE, increasing the loading concentration to 300 μ M vc-MMAE further increased efficacy. Importantly, comparable efficacy was observed between 0 and 1M Gdn conditions at 15mM TCEP at 300. mu.M vc-MMAE; this indicates that loading can be performed in the absence of guanidine hydrochloride as long as the reduction conditions and loading concentration are optimized.

The MMAE experiments demonstrated a complex interaction between the concentrations of TCEP, guanidine hydrochloride and Val-Cit-MMAE and showed many interacting interactions (i.e., 1M Gdn was better than 2M, FIG. 8B; 15mM TCEP was better than 50mM, FIGS. 9A and 9B; increasing the concentration of TCEP from 1.5mM to 15mM increased efficacy in the absence of Gdn but no difference in 1M Gdn, FIG. 9B).

Example 4

Exosome-linked PROTAC

According to fig. 10C, TBK1PROTAC is attached to exosomes according to the method disclosed above. PROTAC comprises a TBK1 targeting ligand, a linker and a ligand capable of binding to VHL E3 ubiquitin ligase.

PROTAC is attached to an exosome, e.g., the outer surface of an exosome membrane and/or a cavity surface, via a maleimide moiety (either directly or indirectly through a linker). ProTAC can be attached to exosomes through a maleimide-VA-PABC cleavable linker. PROTAC can be linked to exosomes through a VHL (E3 ligase) binding ligand.

The function of TBK1PROTAC linked to an exosome of the present disclosure can be determined using in vitro or in vivo methods. In vitro methods include western blotting to (I) directly measure TBK1 degradation in cell lines, (ii) determine inhibition of IRF3 phosphorylation following stimulation with STING agonists (poly I: C, CL656, LPS, etc.), or (iii) determine TBK1 protein knockdown in human monocytes. Another in vitro assay can determine inhibition of STING agonism, for example, using a B16IRF reporter cell line (e.g., pretreated with exosome-PROTAC conjugate, stimulated with STING agonist, and measured for reporter response) or human monocytes (e.g., measured for IFB β release).

In vivo assays for determining the function of the exosome-PROTAC conjugates of the present disclosure include, for example, assays that determine: (i) TBK1 protein knock-down in peritoneal macrophages (e.g., intraperitoneal administration with exosome-PROTAC conjugate, harvesting peritoneal macrophages, and measuring knock-down by western blot or flow cytometry), (ii) inhibition of STING agonist-induced serum cytokines (e.g., intraperitoneal pretreatment with exosome-PROTAC conjugate, intraperitoneal stimulation with STING agonist, and measurement of plasma/serum cytokines at certain time points), or (iii) inhibition of STING agonist-induced phospho IRF3 (e.g., determining pIRF3 levels in different cell types if knock-down is very selective and no reduction in serum cytokines is observed, e.g., using anti-pIRF antibodies and flow cytometry can help show selectivity).

Example 5

Exosome-associated LPA1 inhibitor-exotam 152

Lysophosphatidic acid (LPA) is a highly potent endogenous lipid mediator that protects and rescues cells from programmed cell death. LPA acts as an important mediator of fibrogenesis through the action of its high affinity LPA1 receptor.

AM152 (also known as BMS-986020) is a specific LPA1 inhibitor. AM152 is a high affinity LPA1 antagonist, inhibits bile acid and phospholipid transporters, and has IC for BSEP, MRP4 and MDR3₅₀4.8. mu.M, 6.2. mu.M and 7.5. mu.M, respectively. The chemical structures of LPA1 inhibitors AM152 and AM095 are provided in fig. 12. The figure shows that maleimide-containing reagents can be conjugated to the carboxylic acid and/or carbamate groups of AM 152. Since the same reactive groups are present in AM095, AM095 can be derivatized using the same method.

LPA1 antagonists (such as AM095 and AM152) can be chemically attached to the surface of exosomes using the methods disclosed in the present specification. The result will be that the surface of the EV (e.g., exosome) contains multiple antagonist molecules. See fig. 13.

Figure 14 shows an example of how maleimide reactive groups were added to AM152 via acid groups. This example shows a maleimide group as part of a complex comprising an ala-val cleavable linker interposed between the maleimide group and a carboxylic acid-reactive chloromethylphenyl group. Figure 15 shows two exemplary reagents that can be used to derivatize AM 152. The above reagents comprise (i) a chloromethylphenyl group which can react with the carboxylic acid group of AM152 and (ii) a maleimide group; and interposed between them is a cleavable cit-val dipeptide and a C5 spacer. The following reagents contained (i) a chloromethylphenyl group which could react with the carboxylic acid group of AM152 and (ii) a maleimide group, with a cleavable ala-val dipeptide and a C5 spacer interposed between them. The maleimide group will then be used to link AM152 (or AM095), for example directly or indirectly to a scaffold moiety via one or more spacers or linkers.

FIG. 16 shows the products that would result from cleavage of the cit-val or ala-val dipeptide in the conjugate product (e.g., by cathepsin B). This product AM152 anilino ester can be further processed by endogenous esterases to yield the free acid AM152 product.

Figure 17 shows several AM152 derivatives containing free maleimide groups and different spacer combinations. Additional derivatives are shown in figure 18.

Fig. 19 shows that after protection of the carboxylic acid groups, AM152 can be derivatized at the carbamate groups using the same reagents used to derivatize the carboxylic acid groups. The resulting product is then deprotected to release the carboxylic acid group.

Fig. 20 shows an example in which a complex having a maleimide group is linked to the carbamate group of AM152 via a linker. Suitable linkers include any of the linkers disclosed in this specification.

The process disclosed in this example involves the generation of AM152 or AM095 derivatives comprising a free maleimide reactive group, which linker can then be reacted directly or indirectly with a scaffold moiety through one or more spacers or linkers. As a result, AM152 or AM095 will attach to the outer surface of the EV (e.g., exosomes).

However, the present invention can also be implemented by the following steps: the scaffold moiety is first derivatized, e.g., with a bifunctional group comprising maleimide, and then the derivatized scaffold moiety (e.g., with a free chloromethylphenyl group) is reacted with a carboxylic acid or carbamate group of AM152, as shown in fig. 21.

In some aspects, chemically attaching AM152 (or AM095) to the surface of an EV (e.g., exosome) via a maleimide moiety improves at least one beneficial property of unconjugated AM152 (or AM095) and/or reduces at least one detrimental property (e.g., toxicity, such as gallbladder toxicity and/or liver toxicity) of unconjugated AM152 or AM 095. In some aspects, chemically linking AM152 (or AM095) to an EV (e.g., exosome) via a maleimide moiety improves the efficacy of AM512 or AM095 (as compared to free AM152 or free AM095) in treating fibrotic diseases (e.g., pulmonary fibrosis, such as IPF).

Example 6

Exosome-associated NLRP3 inhibitor-ExoMCC 950

MCC950(N- [ [ (1,2,3,5,6, 7-hexahydro-sym-indenopadiene-4-yl) amino ] carbonyl ] -4- (1-hydroxy-1-methylethyl) -2-furansulfonamide) is a potent and selective inhibitor of the NLRP3 (NOD-like receptor (NLR) pyrin domain containing protein 3) inflammasome. MCC950 blocks the release of IL-1 β induced by NLRP3 activators such as ATP, MSU and nigericin by preventing oligomerization of the inflammatory body adapter protein ASC (CARD-containing apoptosis-related speck-like protein). Coll et al (2015) Nature Med.21: 248-. MCC950 blocks the release of IL-1 β in macrophages, which are primed by LPS and activated by ATP or nigericin with an IC50 of about 7.5 nM. While MCC950 would prevent NLRP 3-induced release of IL-1 β, MCC950 would not inhibit NLRC4, AIM2 or NLRP1 inflammasome. In addition, MCC950 does not inhibit TLR2 signaling or initiation of NLRP 3.

MCC950 is active in vivo, and blocks IL-1 β production and increases survival in a mouse model of multiple sclerosis. MCC950 also inhibits NLRP 3-induced IL-1 β production in a myocardial infarction model. van Hout et al (2015) eur. MCC950 is also active in ex vivo samples from individuals with mookler-wells syndrome. MCC950 is therefore a potential therapeutic agent for the treatment of NLRP 3-related syndromes, including autoinflammatory and autoimmune diseases.

Figure 22 shows the structure of MCC950, bifunctional reagents that can be used to derivatize MCC950 to introduce maleimide reactive groups, and MCC950 derivatives containing maleimide reactive groups. The phenyl group (×) of the bifunctional reagent may react with the carbamate group (×) of MCC950, yielding the MCC950 derivative depicted in figure 22.

The processes disclosed in this example involve the generation of MCC950 derivatives comprising free maleimide reactive groups, and optionally one of a plurality of linkers (e.g., cleavable linkers and/or one or more spacers) interposed between the MCC950 moiety and the maleimide group, which linkers can then be reacted directly or indirectly with the scaffold moiety through the one or more spacers or linkers. As a result, MCC950 will attach to the outer surface of the EV (e.g., exosomes).

The invention can also be implemented by the following steps: the scaffold moiety is first derivatized, for example with a bifunctional group comprising a maleimide, and the derivatized scaffold moiety (e.g., having a free chloromethylphenyl group or a phenyl group) is then reacted with the carbamate group of MCC950 or another suitable derivatizable group.

In some aspects, chemically attaching MCC950 to the surface of an EV (e.g., exosome) via a maleimide moiety improves at least one beneficial property of unconjugated MCC950 and/or reduces at least one detrimental property (e.g., toxicity, such as gallbladder toxicity and/or hepatotoxicity) of unconjugated MCC 950. In some aspects, chemically linking MCC950 to EVs (e.g., exosomes) via a maleimide moiety improves the efficacy of MCC950 (as compared to free MCC 950) in the treatment of NLRP3 inflammasome-related diseases or disorders, such as multiple sclerosis, type 2 diabetes, alzheimer's disease, atherosclerosis, neuroinflammation, parkinson's disease, prion disease, cardiac injury caused by myocardial infarction, gout, and generally any NLRP 3-related syndrome, including autoinflammatory and autoimmune diseases.

***

It is to be understood that the detailed description and not the summary and abstract sections are intended to be used to interpret the claims. The summary and abstract sections of the specification may set forth one or more, but not all exemplary aspects of the invention as contemplated by the inventors, and are therefore not intended to limit the disclosure and appended claims in any way.

The present disclosure has been described above with the aid of functional building blocks illustrating the implementation of specific functions and relationships thereof. Boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

The foregoing description of the specific aspects will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific aspects without departing from the general concept of the present invention. Accordingly, such adaptations and modifications are intended to be within the scope and equivalents of the disclosed aspects, based on the teachings and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.

The contents of all cited references (including references, patents, patent applications, and websites) that may be cited in this application are hereby expressly incorporated by reference in their entirety for any purpose, as are the cited references.

Sequence listing

<110> Kodiaak biosciences Inc

<120> extracellular vesicle conjugate and use thereof

<130> 4000.037PC03

<150> US 62/822,014

<151> 2019-03-21

<150> US 62/835,439

<151> 2019-04-17

<160> 231

<170> PatentIn version 3.5

<210> 1

<211> 879

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 1

Met Gly Arg Leu Ala Ser Arg Pro Leu Leu Leu Ala Leu Leu Ser Leu

1 5 10 15

Ala Leu Cys Arg Gly Arg Val Val Arg Val Pro Thr Ala Thr Leu Val

20 25 30

Arg Val Val Gly Thr Glu Leu Val Ile Pro Cys Asn Val Ser Asp Tyr

35 40 45

Asp Gly Pro Ser Glu Gln Asn Phe Asp Trp Ser Phe Ser Ser Leu Gly

50 55 60

Ser Ser Phe Val Glu Leu Ala Ser Thr Trp Glu Val Gly Phe Pro Ala

65 70 75 80

Gln Leu Tyr Gln Glu Arg Leu Gln Arg Gly Glu Ile Leu Leu Arg Arg

85 90 95

Thr Ala Asn Asp Ala Val Glu Leu His Ile Lys Asn Val Gln Pro Ser

100 105 110

Asp Gln Gly His Tyr Lys Cys Ser Thr Pro Ser Thr Asp Ala Thr Val

115 120 125

Gln Gly Asn Tyr Glu Asp Thr Val Gln Val Lys Val Leu Ala Asp Ser

130 135 140

Leu His Val Gly Pro Ser Ala Arg Pro Pro Pro Ser Leu Ser Leu Arg

145 150 155 160

Glu Gly Glu Pro Phe Glu Leu Arg Cys Thr Ala Ala Ser Ala Ser Pro

165 170 175

Leu His Thr His Leu Ala Leu Leu Trp Glu Val His Arg Gly Pro Ala

180 185 190

Arg Arg Ser Val Leu Ala Leu Thr His Glu Gly Arg Phe His Pro Gly

195 200 205

Leu Gly Tyr Glu Gln Arg Tyr His Ser Gly Asp Val Arg Leu Asp Thr

210 215 220

Val Gly Ser Asp Ala Tyr Arg Leu Ser Val Ser Arg Ala Leu Ser Ala

225 230 235 240

Asp Gln Gly Ser Tyr Arg Cys Ile Val Ser Glu Trp Ile Ala Glu Gln

245 250 255

Gly Asn Trp Gln Glu Ile Gln Glu Lys Ala Val Glu Val Ala Thr Val

260 265 270

Val Ile Gln Pro Ser Val Leu Arg Ala Ala Val Pro Lys Asn Val Ser

275 280 285

Val Ala Glu Gly Lys Glu Leu Asp Leu Thr Cys Asn Ile Thr Thr Asp

290 295 300

Arg Ala Asp Asp Val Arg Pro Glu Val Thr Trp Ser Phe Ser Arg Met

305 310 315 320

Pro Asp Ser Thr Leu Pro Gly Ser Arg Val Leu Ala Arg Leu Asp Arg

325 330 335

Asp Ser Leu Val His Ser Ser Pro His Val Ala Leu Ser His Val Asp

340 345 350

Ala Arg Ser Tyr His Leu Leu Val Arg Asp Val Ser Lys Glu Asn Ser

355 360 365

Gly Tyr Tyr Tyr Cys His Val Ser Leu Trp Ala Pro Gly His Asn Arg

370 375 380

Ser Trp His Lys Val Ala Glu Ala Val Ser Ser Pro Ala Gly Val Gly

385 390 395 400

Val Thr Trp Leu Glu Pro Asp Tyr Gln Val Tyr Leu Asn Ala Ser Lys

405 410 415

Val Pro Gly Phe Ala Asp Asp Pro Thr Glu Leu Ala Cys Arg Val Val

420 425 430

Asp Thr Lys Ser Gly Glu Ala Asn Val Arg Phe Thr Val Ser Trp Tyr

435 440 445

Tyr Arg Met Asn Arg Arg Ser Asp Asn Val Val Thr Ser Glu Leu Leu

450 455 460

Ala Val Met Asp Gly Asp Trp Thr Leu Lys Tyr Gly Glu Arg Ser Lys

465 470 475 480

Gln Arg Ala Gln Asp Gly Asp Phe Ile Phe Ser Lys Glu His Thr Asp

485 490 495

Thr Phe Asn Phe Arg Ile Gln Arg Thr Thr Glu Glu Asp Arg Gly Asn

500 505 510

Tyr Tyr Cys Val Val Ser Ala Trp Thr Lys Gln Arg Asn Asn Ser Trp

515 520 525

Val Lys Ser Lys Asp Val Phe Ser Lys Pro Val Asn Ile Phe Trp Ala

530 535 540

Leu Glu Asp Ser Val Leu Val Val Lys Ala Arg Gln Pro Lys Pro Phe

545 550 555 560

Phe Ala Ala Gly Asn Thr Phe Glu Met Thr Cys Lys Val Ser Ser Lys

565 570 575

Asn Ile Lys Ser Pro Arg Tyr Ser Val Leu Ile Met Ala Glu Lys Pro

580 585 590

Val Gly Asp Leu Ser Ser Pro Asn Glu Thr Lys Tyr Ile Ile Ser Leu

595 600 605

Asp Gln Asp Ser Val Val Lys Leu Glu Asn Trp Thr Asp Ala Ser Arg

610 615 620

Val Asp Gly Val Val Leu Glu Lys Val Gln Glu Asp Glu Phe Arg Tyr

625 630 635 640

Arg Met Tyr Gln Thr Gln Val Ser Asp Ala Gly Leu Tyr Arg Cys Met

645 650 655

Val Thr Ala Trp Ser Pro Val Arg Gly Ser Leu Trp Arg Glu Ala Ala

660 665 670

Thr Ser Leu Ser Asn Pro Ile Glu Ile Asp Phe Gln Thr Ser Gly Pro

675 680 685

Ile Phe Asn Ala Ser Val His Ser Asp Thr Pro Ser Val Ile Arg Gly

690 695 700

Asp Leu Ile Lys Leu Phe Cys Ile Ile Thr Val Glu Gly Ala Ala Leu

705 710 715 720

Asp Pro Asp Asp Met Ala Phe Asp Val Ser Trp Phe Ala Val His Ser

725 730 735

Phe Gly Leu Asp Lys Ala Pro Val Leu Leu Ser Ser Leu Asp Arg Lys

740 745 750

Gly Ile Val Thr Thr Ser Arg Arg Asp Trp Lys Ser Asp Leu Ser Leu

755 760 765

Glu Arg Val Ser Val Leu Glu Phe Leu Leu Gln Val His Gly Ser Glu

770 775 780

Asp Gln Asp Phe Gly Asn Tyr Tyr Cys Ser Val Thr Pro Trp Val Lys

785 790 795 800

Ser Pro Thr Gly Ser Trp Gln Lys Glu Ala Glu Ile His Ser Lys Pro

805 810 815

Val Phe Ile Thr Val Lys Met Asp Val Leu Asn Ala Phe Lys Tyr Pro

820 825 830

Leu Leu Ile Gly Val Gly Leu Ser Thr Val Ile Gly Leu Leu Ser Cys

835 840 845

Leu Ile Gly Tyr Cys Ser Ser His Trp Cys Cys Lys Lys Glu Val Gln

850 855 860

Glu Thr Arg Arg Glu Arg Arg Arg Leu Met Ser Met Glu Met Asp

865 870 875

<210> 2

<211> 192

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 2

Gly Pro Ile Phe Asn Ala Ser Val His Ser Asp Thr Pro Ser Val Ile

1 5 10 15

Arg Gly Asp Leu Ile Lys Leu Phe Cys Ile Ile Thr Val Glu Gly Ala

20 25 30

Ala Leu Asp Pro Asp Asp Met Ala Phe Asp Val Ser Trp Phe Ala Val

35 40 45

His Ser Phe Gly Leu Asp Lys Ala Pro Val Leu Leu Ser Ser Leu Asp

50 55 60

Arg Lys Gly Ile Val Thr Thr Ser Arg Arg Asp Trp Lys Ser Asp Leu

65 70 75 80

Ser Leu Glu Arg Val Ser Val Leu Glu Phe Leu Leu Gln Val His Gly

85 90 95

Ser Glu Asp Gln Asp Phe Gly Asn Tyr Tyr Cys Ser Val Thr Pro Trp

100 105 110

Val Lys Ser Pro Thr Gly Ser Trp Gln Lys Glu Ala Glu Ile His Ser

115 120 125

Lys Pro Val Phe Ile Thr Val Lys Met Asp Val Leu Asn Ala Phe Lys

130 135 140

Tyr Pro Leu Leu Ile Gly Val Gly Leu Ser Thr Val Ile Gly Leu Leu

145 150 155 160

Ser Cys Leu Ile Gly Tyr Cys Ser Ser His Trp Cys Cys Lys Lys Glu

165 170 175

Val Gln Glu Thr Arg Arg Glu Arg Arg Arg Leu Met Ser Met Glu Met

180 185 190

<210> 3

<211> 385

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 3

Met Ala Ala Ala Leu Phe Val Leu Leu Gly Phe Ala Leu Leu Gly Thr

1 5 10 15

His Gly Ala Ser Gly Ala Ala Gly Phe Val Gln Ala Pro Leu Ser Gln

20 25 30

Gln Arg Trp Val Gly Gly Ser Val Glu Leu His Cys Glu Ala Val Gly

35 40 45

Ser Pro Val Pro Glu Ile Gln Trp Trp Phe Glu Gly Gln Gly Pro Asn

50 55 60

Asp Thr Cys Ser Gln Leu Trp Asp Gly Ala Arg Leu Asp Arg Val His

65 70 75 80

Ile His Ala Thr Tyr His Gln His Ala Ala Ser Thr Ile Ser Ile Asp

85 90 95

Thr Leu Val Glu Glu Asp Thr Gly Thr Tyr Glu Cys Arg Ala Ser Asn

100 105 110

Asp Pro Asp Arg Asn His Leu Thr Arg Ala Pro Arg Val Lys Trp Val

115 120 125

Arg Ala Gln Ala Val Val Leu Val Leu Glu Pro Gly Thr Val Phe Thr

130 135 140

Thr Val Glu Asp Leu Gly Ser Lys Ile Leu Leu Thr Cys Ser Leu Asn

145 150 155 160

Asp Ser Ala Thr Glu Val Thr Gly His Arg Trp Leu Lys Gly Gly Val

165 170 175

Val Leu Lys Glu Asp Ala Leu Pro Gly Gln Lys Thr Glu Phe Lys Val

180 185 190

Asp Ser Asp Asp Gln Trp Gly Glu Tyr Ser Cys Val Phe Leu Pro Glu

195 200 205

Pro Met Gly Thr Ala Asn Ile Gln Leu His Gly Pro Pro Arg Val Lys

210 215 220

Ala Val Lys Ser Ser Glu His Ile Asn Glu Gly Glu Thr Ala Met Leu

225 230 235 240

Val Cys Lys Ser Glu Ser Val Pro Pro Val Thr Asp Trp Ala Trp Tyr

245 250 255

Lys Ile Thr Asp Ser Glu Asp Lys Ala Leu Met Asn Gly Ser Glu Ser

260 265 270

Arg Phe Phe Val Ser Ser Ser Gln Gly Arg Ser Glu Leu His Ile Glu

275 280 285

Asn Leu Asn Met Glu Ala Asp Pro Gly Gln Tyr Arg Cys Asn Gly Thr

290 295 300

Ser Ser Lys Gly Ser Asp Gln Ala Ile Ile Thr Leu Arg Val Arg Ser

305 310 315 320

His Leu Ala Ala Leu Trp Pro Phe Leu Gly Ile Val Ala Glu Val Leu

325 330 335

Val Leu Val Thr Ile Ile Phe Ile Tyr Glu Lys Arg Arg Lys Pro Glu

340 345 350

Asp Val Leu Asp Asp Asp Asp Ala Gly Ser Ala Pro Leu Lys Ser Ser

355 360 365

Gly Gln His Gln Asn Asp Lys Gly Lys Asn Val Arg Gln Arg Asn Ser

370 375 380

Ser

385

<210> 4

<211> 613

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 4

Met Gly Ala Leu Arg Pro Thr Leu Leu Pro Pro Ser Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Met Leu Gly Met Gly Cys Trp Ala Arg Glu Val Leu Val

20 25 30

Pro Glu Gly Pro Leu Tyr Arg Val Ala Gly Thr Ala Val Ser Ile Ser

35 40 45

Cys Asn Val Thr Gly Tyr Glu Gly Pro Ala Gln Gln Asn Phe Glu Trp

50 55 60

Phe Leu Tyr Arg Pro Glu Ala Pro Asp Thr Ala Leu Gly Ile Val Ser

65 70 75 80

Thr Lys Asp Thr Gln Phe Ser Tyr Ala Val Phe Lys Ser Arg Val Val

85 90 95

Ala Gly Glu Val Gln Val Gln Arg Leu Gln Gly Asp Ala Val Val Leu

100 105 110

Lys Ile Ala Arg Leu Gln Ala Gln Asp Ala Gly Ile Tyr Glu Cys His

115 120 125

Thr Pro Ser Thr Asp Thr Arg Tyr Leu Gly Ser Tyr Ser Gly Lys Val

130 135 140

Glu Leu Arg Val Leu Pro Asp Val Leu Gln Val Ser Ala Ala Pro Pro

145 150 155 160

Gly Pro Arg Gly Arg Gln Ala Pro Thr Ser Pro Pro Arg Met Thr Val

165 170 175

His Glu Gly Gln Glu Leu Ala Leu Gly Cys Leu Ala Arg Thr Ser Thr

180 185 190

Gln Lys His Thr His Leu Ala Val Ser Phe Gly Arg Ser Val Pro Glu

195 200 205

Ala Pro Val Gly Arg Ser Thr Leu Gln Glu Val Val Gly Ile Arg Ser

210 215 220

Asp Leu Ala Val Glu Ala Gly Ala Pro Tyr Ala Glu Arg Leu Ala Ala

225 230 235 240

Gly Glu Leu Arg Leu Gly Lys Glu Gly Thr Asp Arg Tyr Arg Met Val

245 250 255

Val Gly Gly Ala Gln Ala Gly Asp Ala Gly Thr Tyr His Cys Thr Ala

260 265 270

Ala Glu Trp Ile Gln Asp Pro Asp Gly Ser Trp Ala Gln Ile Ala Glu

275 280 285

Lys Arg Ala Val Leu Ala His Val Asp Val Gln Thr Leu Ser Ser Gln

290 295 300

Leu Ala Val Thr Val Gly Pro Gly Glu Arg Arg Ile Gly Pro Gly Glu

305 310 315 320

Pro Leu Glu Leu Leu Cys Asn Val Ser Gly Ala Leu Pro Pro Ala Gly

325 330 335

Arg His Ala Ala Tyr Ser Val Gly Trp Glu Met Ala Pro Ala Gly Ala

340 345 350

Pro Gly Pro Gly Arg Leu Val Ala Gln Leu Asp Thr Glu Gly Val Gly

355 360 365

Ser Leu Gly Pro Gly Tyr Glu Gly Arg His Ile Ala Met Glu Lys Val

370 375 380

Ala Ser Arg Thr Tyr Arg Leu Arg Leu Glu Ala Ala Arg Pro Gly Asp

385 390 395 400

Ala Gly Thr Tyr Arg Cys Leu Ala Lys Ala Tyr Val Arg Gly Ser Gly

405 410 415

Thr Arg Leu Arg Glu Ala Ala Ser Ala Arg Ser Arg Pro Leu Pro Val

420 425 430

His Val Arg Glu Glu Gly Val Val Leu Glu Ala Val Ala Trp Leu Ala

435 440 445

Gly Gly Thr Val Tyr Arg Gly Glu Thr Ala Ser Leu Leu Cys Asn Ile

450 455 460

Ser Val Arg Gly Gly Pro Pro Gly Leu Arg Leu Ala Ala Ser Trp Trp

465 470 475 480

Val Glu Arg Pro Glu Asp Gly Glu Leu Ser Ser Val Pro Ala Gln Leu

485 490 495

Val Gly Gly Val Gly Gln Asp Gly Val Ala Glu Leu Gly Val Arg Pro

500 505 510

Gly Gly Gly Pro Val Ser Val Glu Leu Val Gly Pro Arg Ser His Arg

515 520 525

Leu Arg Leu His Ser Leu Gly Pro Glu Asp Glu Gly Val Tyr His Cys

530 535 540

Ala Pro Ser Ala Trp Val Gln His Ala Asp Tyr Ser Trp Tyr Gln Ala

545 550 555 560

Gly Ser Ala Arg Ser Gly Pro Val Thr Val Tyr Pro Tyr Met His Ala

565 570 575

Leu Asp Thr Leu Phe Val Pro Leu Leu Val Gly Thr Gly Val Ala Leu

580 585 590

Val Thr Gly Ala Thr Val Leu Gly Thr Ile Thr Cys Cys Phe Met Lys

595 600 605

Arg Leu Arg Lys Arg

610

<210> 5

<211> 748

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 5

Met Asn Leu Gln Pro Ile Phe Trp Ile Gly Leu Ile Ser Ser Val Cys

1 5 10 15

Cys Val Phe Ala Gln Thr Asp Glu Asn Arg Cys Leu Lys Ala Asn Ala

20 25 30

Lys Ser Cys Gly Glu Cys Ile Gln Ala Gly Pro Asn Cys Gly Trp Cys

35 40 45

Thr Asn Ser Thr Phe Leu Gln Glu Gly Met Pro Thr Ser Ala Arg Cys

50 55 60

Asp Asp Leu Glu Ala Leu Lys Lys Lys Gly Cys Pro Pro Asp Asp Ile

65 70 75 80

Glu Asn Pro Arg Gly Ser Lys Asp Ile Lys Lys Asn Lys Asn Val Thr

85 90 95

Asn Arg Ser Lys Gly Thr Ala Glu Lys Leu Lys Pro Glu Asp Ile Thr

100 105 110

Gln Ile Gln Pro Gln Gln Leu Val Leu Arg Leu Arg Ser Gly Glu Pro

115 120 125

Gln Thr Phe Thr Leu Lys Phe Lys Arg Ala Glu Asp Tyr Pro Ile Asp

130 135 140

Leu Tyr Tyr Leu Met Asp Leu Ser Tyr Ser Met Lys Asp Asp Leu Glu

145 150 155 160

Asn Val Lys Ser Leu Gly Thr Asp Leu Met Asn Glu Met Arg Arg Ile

165 170 175

Thr Ser Asp Phe Arg Ile Gly Phe Gly Ser Phe Val Glu Lys Thr Val

180 185 190

Met Pro Tyr Ile Ser Thr Thr Pro Ala Lys Leu Arg Asn Pro Cys Thr

195 200 205

Ser Glu Gln Asn Cys Thr Ser Pro Phe Ser Tyr Lys Asn Val Leu Ser

210 215 220

Leu Thr Asn Lys Gly Glu Val Phe Asn Glu Leu Val Gly Lys Gln Arg

225 230 235 240

Ile Ser Gly Asn Leu Asp Ser Pro Glu Gly Gly Phe Asp Ala Ile Met

245 250 255

Gln Val Ala Val Cys Gly Ser Leu Ile Gly Trp Arg Asn Val Thr Arg

260 265 270

Leu Leu Val Phe Ser Thr Asp Ala Gly Phe His Phe Ala Gly Asp Gly

275 280 285

Lys Leu Gly Gly Ile Val Leu Pro Asn Asp Gly Gln Cys His Leu Glu

290 295 300

Asn Asn Met Tyr Thr Met Ser His Tyr Tyr Asp Tyr Pro Ser Ile Ala

305 310 315 320

His Leu Val Gln Lys Leu Ser Glu Asn Asn Ile Gln Thr Ile Phe Ala

325 330 335

Val Thr Glu Glu Phe Gln Pro Val Tyr Lys Glu Leu Lys Asn Leu Ile

340 345 350

Pro Lys Ser Ala Val Gly Thr Leu Ser Ala Asn Ser Ser Asn Val Ile

355 360 365

Gln Leu Ile Ile Asp Ala Tyr Asn Ser Leu Ser Ser Glu Val Ile Leu

370 375 380

Glu Asn Gly Lys Leu Ser Glu Gly Val Thr Ile Ser Tyr Lys Ser Tyr

385 390 395 400

Cys Lys Asn Gly Val Asn Gly Thr Gly Glu Asn Gly Arg Lys Cys Ser

405 410 415

Asn Ile Ser Ile Gly Asp Glu Val Gln Phe Glu Ile Ser Ile Thr Ser

420 425 430

Asn Lys Cys Pro Lys Lys Asp Ser Asp Ser Phe Lys Ile Arg Pro Leu

435 440 445

Gly Phe Thr Glu Glu Val Glu Val Ile Leu Gln Tyr Ile Cys Glu Cys

450 455 460

Glu Cys Gln Ser Glu Gly Ile Pro Glu Ser Pro Lys Cys His Glu Gly

465 470 475 480

Asn Gly Thr Phe Glu Cys Gly Ala Cys Arg Cys Asn Glu Gly Arg Val

485 490 495

Gly Arg His Cys Glu Cys Ser Thr Asp Glu Val Asn Ser Glu Asp Met

500 505 510

Asp Ala Tyr Cys Arg Lys Glu Asn Ser Ser Glu Ile Cys Ser Asn Asn

515 520 525

Gly Glu Cys Val Cys Gly Gln Cys Val Cys Arg Lys Arg Asp Asn Thr

530 535 540

Asn Glu Ile Tyr Ser Gly Ala Ser Asn Gly Gln Ile Cys Asn Gly Arg

545 550 555 560

Gly Ile Cys Glu Cys Gly Val Cys Lys Cys Thr Asp Pro Lys Phe Gln

565 570 575

Gly Gln Thr Cys Glu Met Cys Gln Thr Cys Leu Gly Val Cys Ala Glu

580 585 590

His Lys Glu Cys Val Gln Cys Arg Ala Phe Asn Lys Gly Glu Lys Lys

595 600 605

Asp Thr Cys Thr Gln Glu Cys Ser Tyr Phe Asn Ile Thr Lys Val Glu

610 615 620

Ser Arg Asp Lys Leu Pro Gln Pro Val Gln Pro Asp Pro Val Ser His

625 630 635 640

Cys Lys Glu Lys Asp Val Asp Asp Cys Trp Phe Tyr Phe Thr Tyr Ser

645 650 655

Val Asn Gly Asn Asn Glu Val Met Val His Val Val Glu Asn Pro Glu

660 665 670

Cys Pro Thr Gly Pro Asp Ile Ile Pro Ile Val Ala Gly Val Val Ala

675 680 685

Gly Ile Val Leu Ile Gly Leu Ala Leu Leu Leu Ile Trp Lys Leu Leu

690 695 700

Met Ile Ile His Asp Arg Arg Glu Phe Ala Lys Phe Glu Lys Glu Lys

705 710 715 720

Met Asn Ala Lys Trp Asp Thr Gly Glu Asn Pro Ile Tyr Lys Ser Ala

725 730 735

Val Thr Thr Val Val Asn Pro Lys Tyr Glu Gly Lys

740 745

<210> 6

<211> 1032

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 6

Met Ala Trp Glu Ala Arg Arg Glu Pro Gly Pro Arg Arg Ala Ala Val

1 5 10 15

Arg Glu Thr Val Met Leu Leu Leu Cys Leu Gly Val Pro Thr Gly Arg

20 25 30

Pro Tyr Asn Val Asp Thr Glu Ser Ala Leu Leu Tyr Gln Gly Pro His

35 40 45

Asn Thr Leu Phe Gly Tyr Ser Val Val Leu His Ser His Gly Ala Asn

50 55 60

Arg Trp Leu Leu Val Gly Ala Pro Thr Ala Asn Trp Leu Ala Asn Ala

65 70 75 80

Ser Val Ile Asn Pro Gly Ala Ile Tyr Arg Cys Arg Ile Gly Lys Asn

85 90 95

Pro Gly Gln Thr Cys Glu Gln Leu Gln Leu Gly Ser Pro Asn Gly Glu

100 105 110

Pro Cys Gly Lys Thr Cys Leu Glu Glu Arg Asp Asn Gln Trp Leu Gly

115 120 125

Val Thr Leu Ser Arg Gln Pro Gly Glu Asn Gly Ser Ile Val Thr Cys

130 135 140

Gly His Arg Trp Lys Asn Ile Phe Tyr Ile Lys Asn Glu Asn Lys Leu

145 150 155 160

Pro Thr Gly Gly Cys Tyr Gly Val Pro Pro Asp Leu Arg Thr Glu Leu

165 170 175

Ser Lys Arg Ile Ala Pro Cys Tyr Gln Asp Tyr Val Lys Lys Phe Gly

180 185 190

Glu Asn Phe Ala Ser Cys Gln Ala Gly Ile Ser Ser Phe Tyr Thr Lys

195 200 205

Asp Leu Ile Val Met Gly Ala Pro Gly Ser Ser Tyr Trp Thr Gly Ser

210 215 220

Leu Phe Val Tyr Asn Ile Thr Thr Asn Lys Tyr Lys Ala Phe Leu Asp

225 230 235 240

Lys Gln Asn Gln Val Lys Phe Gly Ser Tyr Leu Gly Tyr Ser Val Gly

245 250 255

Ala Gly His Phe Arg Ser Gln His Thr Thr Glu Val Val Gly Gly Ala

260 265 270

Pro Gln His Glu Gln Ile Gly Lys Ala Tyr Ile Phe Ser Ile Asp Glu

275 280 285

Lys Glu Leu Asn Ile Leu His Glu Met Lys Gly Lys Lys Leu Gly Ser

290 295 300

Tyr Phe Gly Ala Ser Val Cys Ala Val Asp Leu Asn Ala Asp Gly Phe

305 310 315 320

Ser Asp Leu Leu Val Gly Ala Pro Met Gln Ser Thr Ile Arg Glu Glu

325 330 335

Gly Arg Val Phe Val Tyr Ile Asn Ser Gly Ser Gly Ala Val Met Asn

340 345 350

Ala Met Glu Thr Asn Leu Val Gly Ser Asp Lys Tyr Ala Ala Arg Phe

355 360 365

Gly Glu Ser Ile Val Asn Leu Gly Asp Ile Asp Asn Asp Gly Phe Glu

370 375 380

Asp Val Ala Ile Gly Ala Pro Gln Glu Asp Asp Leu Gln Gly Ala Ile

385 390 395 400

Tyr Ile Tyr Asn Gly Arg Ala Asp Gly Ile Ser Ser Thr Phe Ser Gln

405 410 415

Arg Ile Glu Gly Leu Gln Ile Ser Lys Ser Leu Ser Met Phe Gly Gln

420 425 430

Ser Ile Ser Gly Gln Ile Asp Ala Asp Asn Asn Gly Tyr Val Asp Val

435 440 445

Ala Val Gly Ala Phe Arg Ser Asp Ser Ala Val Leu Leu Arg Thr Arg

450 455 460

Pro Val Val Ile Val Asp Ala Ser Leu Ser His Pro Glu Ser Val Asn

465 470 475 480

Arg Thr Lys Phe Asp Cys Val Glu Asn Gly Trp Pro Ser Val Cys Ile

485 490 495

Asp Leu Thr Leu Cys Phe Ser Tyr Lys Gly Lys Glu Val Pro Gly Tyr

500 505 510

Ile Val Leu Phe Tyr Asn Met Ser Leu Asp Val Asn Arg Lys Ala Glu

515 520 525

Ser Pro Pro Arg Phe Tyr Phe Ser Ser Asn Gly Thr Ser Asp Val Ile

530 535 540

Thr Gly Ser Ile Gln Val Ser Ser Arg Glu Ala Asn Cys Arg Thr His

545 550 555 560

Gln Ala Phe Met Arg Lys Asp Val Arg Asp Ile Leu Thr Pro Ile Gln

565 570 575

Ile Glu Ala Ala Tyr His Leu Gly Pro His Val Ile Ser Lys Arg Ser

580 585 590

Thr Glu Glu Phe Pro Pro Leu Gln Pro Ile Leu Gln Gln Lys Lys Glu

595 600 605

Lys Asp Ile Met Lys Lys Thr Ile Asn Phe Ala Arg Phe Cys Ala His

610 615 620

Glu Asn Cys Ser Ala Asp Leu Gln Val Ser Ala Lys Ile Gly Phe Leu

625 630 635 640

Lys Pro His Glu Asn Lys Thr Tyr Leu Ala Val Gly Ser Met Lys Thr

645 650 655

Leu Met Leu Asn Val Ser Leu Phe Asn Ala Gly Asp Asp Ala Tyr Glu

660 665 670

Thr Thr Leu His Val Lys Leu Pro Val Gly Leu Tyr Phe Ile Lys Ile

675 680 685

Leu Glu Leu Glu Glu Lys Gln Ile Asn Cys Glu Val Thr Asp Asn Ser

690 695 700

Gly Val Val Gln Leu Asp Cys Ser Ile Gly Tyr Ile Tyr Val Asp His

705 710 715 720

Leu Ser Arg Ile Asp Ile Ser Phe Leu Leu Asp Val Ser Ser Leu Ser

725 730 735

Arg Ala Glu Glu Asp Leu Ser Ile Thr Val His Ala Thr Cys Glu Asn

740 745 750

Glu Glu Glu Met Asp Asn Leu Lys His Ser Arg Val Thr Val Ala Ile

755 760 765

Pro Leu Lys Tyr Glu Val Lys Leu Thr Val His Gly Phe Val Asn Pro

770 775 780

Thr Ser Phe Val Tyr Gly Ser Asn Asp Glu Asn Glu Pro Glu Thr Cys

785 790 795 800

Met Val Glu Lys Met Asn Leu Thr Phe His Val Ile Asn Thr Gly Asn

805 810 815

Ser Met Ala Pro Asn Val Ser Val Glu Ile Met Val Pro Asn Ser Phe

820 825 830

Ser Pro Gln Thr Asp Lys Leu Phe Asn Ile Leu Asp Val Gln Thr Thr

835 840 845

Thr Gly Glu Cys His Phe Glu Asn Tyr Gln Arg Val Cys Ala Leu Glu

850 855 860

Gln Gln Lys Ser Ala Met Gln Thr Leu Lys Gly Ile Val Arg Phe Leu

865 870 875 880

Ser Lys Thr Asp Lys Arg Leu Leu Tyr Cys Ile Lys Ala Asp Pro His

885 890 895

Cys Leu Asn Phe Leu Cys Asn Phe Gly Lys Met Glu Ser Gly Lys Glu

900 905 910

Ala Ser Val His Ile Gln Leu Glu Gly Arg Pro Ser Ile Leu Glu Met

915 920 925

Asp Glu Thr Ser Ala Leu Lys Phe Glu Ile Arg Ala Thr Gly Phe Pro

930 935 940

Glu Pro Asn Pro Arg Val Ile Glu Leu Asn Lys Asp Glu Asn Val Ala

945 950 955 960

His Val Leu Leu Glu Gly Leu His His Gln Arg Pro Lys Arg Tyr Phe

965 970 975

Thr Ile Val Ile Ile Ser Ser Ser Leu Leu Leu Gly Leu Ile Val Leu

980 985 990

Leu Leu Ile Ser Tyr Val Met Trp Lys Ala Gly Phe Phe Lys Arg Gln

995 1000 1005

Tyr Lys Ser Ile Leu Gln Glu Glu Asn Arg Arg Asp Ser Trp Ser

1010 1015 1020

Tyr Ile Asn Ser Lys Ser Asn Asp Asp

1025 1030

<210> 7

<211> 630

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 7

Met Glu Leu Gln Pro Pro Glu Ala Ser Ile Ala Val Val Ser Ile Pro

1 5 10 15

Arg Gln Leu Pro Gly Ser His Ser Glu Ala Gly Val Gln Gly Leu Ser

20 25 30

Ala Gly Asp Asp Ser Glu Leu Gly Ser His Cys Val Ala Gln Thr Gly

35 40 45

Leu Glu Leu Leu Ala Ser Gly Asp Pro Leu Pro Ser Ala Ser Gln Asn

50 55 60

Ala Glu Met Ile Glu Thr Gly Ser Asp Cys Val Thr Gln Ala Gly Leu

65 70 75 80

Gln Leu Leu Ala Ser Ser Asp Pro Pro Ala Leu Ala Ser Lys Asn Ala

85 90 95

Glu Val Thr Gly Thr Met Ser Gln Asp Thr Glu Val Asp Met Lys Glu

100 105 110

Val Glu Leu Asn Glu Leu Glu Pro Glu Lys Gln Pro Met Asn Ala Ala

115 120 125

Ser Gly Ala Ala Met Ser Leu Ala Gly Ala Glu Lys Asn Gly Leu Val

130 135 140

Lys Ile Lys Val Ala Glu Asp Glu Ala Glu Ala Ala Ala Ala Ala Lys

145 150 155 160

Phe Thr Gly Leu Ser Lys Glu Glu Leu Leu Lys Val Ala Gly Ser Pro

165 170 175

Gly Trp Val Arg Thr Arg Trp Ala Leu Leu Leu Leu Phe Trp Leu Gly

180 185 190

Trp Leu Gly Met Leu Ala Gly Ala Val Val Ile Ile Val Arg Ala Pro

195 200 205

Arg Cys Arg Glu Leu Pro Ala Gln Lys Trp Trp His Thr Gly Ala Leu

210 215 220

Tyr Arg Ile Gly Asp Leu Gln Ala Phe Gln Gly His Gly Ala Gly Asn

225 230 235 240

Leu Ala Gly Leu Lys Gly Arg Leu Asp Tyr Leu Ser Ser Leu Lys Val

245 250 255

Lys Gly Leu Val Leu Gly Pro Ile His Lys Asn Gln Lys Asp Asp Val

260 265 270

Ala Gln Thr Asp Leu Leu Gln Ile Asp Pro Asn Phe Gly Ser Lys Glu

275 280 285

Asp Phe Asp Ser Leu Leu Gln Ser Ala Lys Lys Lys Ser Ile Arg Val

290 295 300

Ile Leu Asp Leu Thr Pro Asn Tyr Arg Gly Glu Asn Ser Trp Phe Ser

305 310 315 320

Thr Gln Val Asp Thr Val Ala Thr Lys Val Lys Asp Ala Leu Glu Phe

325 330 335

Trp Leu Gln Ala Gly Val Asp Gly Phe Gln Val Arg Asp Ile Glu Asn

340 345 350

Leu Lys Asp Ala Ser Ser Phe Leu Ala Glu Trp Gln Asn Ile Thr Lys

355 360 365

Gly Phe Ser Glu Asp Arg Leu Leu Ile Ala Gly Thr Asn Ser Ser Asp

370 375 380

Leu Gln Gln Ile Leu Ser Leu Leu Glu Ser Asn Lys Asp Leu Leu Leu

385 390 395 400

Thr Ser Ser Tyr Leu Ser Asp Ser Gly Ser Thr Gly Glu His Thr Lys

405 410 415

Ser Leu Val Thr Gln Tyr Leu Asn Ala Thr Gly Asn Arg Trp Cys Ser

420 425 430

Trp Ser Leu Ser Gln Ala Arg Leu Leu Thr Ser Phe Leu Pro Ala Gln

435 440 445

Leu Leu Arg Leu Tyr Gln Leu Met Leu Phe Thr Leu Pro Gly Thr Pro

450 455 460

Val Phe Ser Tyr Gly Asp Glu Ile Gly Leu Asp Ala Ala Ala Leu Pro

465 470 475 480

Gly Gln Pro Met Glu Ala Pro Val Met Leu Trp Asp Glu Ser Ser Phe

485 490 495

Pro Asp Ile Pro Gly Ala Val Ser Ala Asn Met Thr Val Lys Gly Gln

500 505 510

Ser Glu Asp Pro Gly Ser Leu Leu Ser Leu Phe Arg Arg Leu Ser Asp

515 520 525

Gln Arg Ser Lys Glu Arg Ser Leu Leu His Gly Asp Phe His Ala Phe

530 535 540

Ser Ala Gly Pro Gly Leu Phe Ser Tyr Ile Arg His Trp Asp Gln Asn

545 550 555 560

Glu Arg Phe Leu Val Val Leu Asn Phe Gly Asp Val Gly Leu Ser Ala

565 570 575

Gly Leu Gln Ala Ser Asp Leu Pro Ala Ser Ala Ser Leu Pro Ala Lys

580 585 590

Ala Asp Leu Leu Leu Ser Thr Gln Pro Gly Arg Glu Glu Gly Ser Pro

595 600 605

Leu Glu Leu Glu Arg Leu Lys Leu Glu Pro His Glu Gly Leu Leu Leu

610 615 620

Arg Phe Pro Tyr Ala Ala

625 630

<210> 8

<211> 332

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 8

Met Gly Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala

1 5 10 15

Glu Arg Pro Gly Glu Ala Ala Val Ala Ser Ser Pro Ser Lys Ala Asn

20 25 30

Gly Gln Glu Asn Gly His Val Lys Val Asn Gly Asp Ala Ser Pro Ala

35 40 45

Ala Ala Glu Ser Gly Ala Lys Glu Glu Leu Gln Ala Asn Gly Ser Ala

50 55 60

Pro Ala Ala Asp Lys Glu Glu Pro Ala Ala Ala Gly Ser Gly Ala Ala

65 70 75 80

Ser Pro Ser Ala Ala Glu Lys Gly Glu Pro Ala Ala Ala Ala Ala Pro

85 90 95

Glu Ala Gly Ala Ser Pro Val Glu Lys Glu Ala Pro Ala Glu Gly Glu

100 105 110

Ala Ala Glu Pro Gly Ser Pro Thr Ala Ala Glu Gly Glu Ala Ala Ser

115 120 125

Ala Ala Ser Ser Thr Ser Ser Pro Lys Ala Glu Asp Gly Ala Thr Pro

130 135 140

Ser Pro Ser Asn Glu Thr Pro Lys Lys Lys Lys Lys Arg Phe Ser Phe

145 150 155 160

Lys Lys Ser Phe Lys Leu Ser Gly Phe Ser Phe Lys Lys Asn Lys Lys

165 170 175

Glu Ala Gly Glu Gly Gly Glu Ala Glu Ala Pro Ala Ala Glu Gly Gly

180 185 190

Lys Asp Glu Ala Ala Gly Gly Ala Ala Ala Ala Ala Ala Glu Ala Gly

195 200 205

Ala Ala Ser Gly Glu Gln Ala Ala Ala Pro Gly Glu Glu Ala Ala Ala

210 215 220

Gly Glu Glu Gly Ala Ala Gly Gly Asp Pro Gln Glu Ala Lys Pro Gln

225 230 235 240

Glu Ala Ala Val Ala Pro Glu Lys Pro Pro Ala Ser Asp Glu Thr Lys

245 250 255

Ala Ala Glu Glu Pro Ser Lys Val Glu Glu Lys Lys Ala Glu Glu Ala

260 265 270

Gly Ala Ser Ala Ala Ala Cys Glu Ala Pro Ser Ala Ala Gly Pro Gly

275 280 285

Ala Pro Pro Glu Gln Glu Ala Ala Pro Ala Glu Glu Pro Ala Ala Ala

290 295 300

Ala Ala Ser Ser Ala Cys Ala Ala Pro Ser Gln Glu Ala Gln Pro Glu

305 310 315 320

Cys Ser Pro Glu Ala Pro Pro Ala Glu Ala Ala Glu

325 330

<210> 9

<211> 195

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 9

Met Gly Ser Gln Ser Ser Lys Ala Pro Arg Gly Asp Val Thr Ala Glu

1 5 10 15

Glu Ala Ala Gly Ala Ser Pro Ala Lys Ala Asn Gly Gln Glu Asn Gly

20 25 30

His Val Lys Ser Asn Gly Asp Leu Ser Pro Lys Gly Glu Gly Glu Ser

35 40 45

Pro Pro Val Asn Gly Thr Asp Glu Ala Ala Gly Ala Thr Gly Asp Ala

50 55 60

Ile Glu Pro Ala Pro Pro Ser Gln Gly Ala Glu Ala Lys Gly Glu Val

65 70 75 80

Pro Pro Lys Glu Thr Pro Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys

85 90 95

Pro Phe Lys Leu Ser Gly Leu Ser Phe Lys Arg Asn Arg Lys Glu Gly

100 105 110

Gly Gly Asp Ser Ser Ala Ser Ser Pro Thr Glu Glu Glu Gln Glu Gln

115 120 125

Gly Glu Ile Gly Ala Cys Ser Asp Glu Gly Thr Ala Gln Glu Gly Lys

130 135 140

Ala Ala Ala Thr Pro Glu Ser Gln Glu Pro Gln Ala Lys Gly Ala Glu

145 150 155 160

Ala Ser Ala Ala Ser Glu Glu Glu Ala Gly Pro Gln Ala Thr Glu Pro

165 170 175

Ser Thr Pro Ser Gly Pro Glu Ser Gly Pro Thr Pro Ala Ser Ala Glu

180 185 190

Gln Asn Glu

195

<210> 10

<211> 227

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 10

Met Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp

1 5 10 15

Glu Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly Ala Ala Thr Glu

20 25 30

Glu Glu Gly Thr Pro Lys Glu Ser Glu Pro Gln Ala Ala Ala Glu Pro

35 40 45

Ala Glu Ala Lys Glu Gly Lys Glu Lys Pro Asp Gln Asp Ala Glu Gly

50 55 60

Lys Ala Glu Glu Lys Glu Gly Glu Lys Asp Ala Ala Ala Ala Lys Glu

65 70 75 80

Glu Ala Pro Lys Ala Glu Pro Glu Lys Thr Glu Gly Ala Ala Glu Ala

85 90 95

Lys Ala Glu Pro Pro Lys Ala Pro Glu Gln Glu Gln Ala Ala Pro Gly

100 105 110

Pro Ala Ala Gly Gly Glu Ala Pro Lys Ala Ala Glu Ala Ala Ala Ala

115 120 125

Pro Ala Glu Ser Ala Ala Pro Ala Ala Gly Glu Glu Pro Ser Lys Glu

130 135 140

Glu Gly Glu Pro Lys Lys Thr Glu Ala Pro Ala Ala Pro Ala Ala Gln

145 150 155 160

Glu Thr Lys Ser Asp Gly Ala Pro Ala Ser Asp Ser Lys Pro Gly Ser

165 170 175

Ser Glu Ala Ala Pro Ser Ser Lys Glu Thr Pro Ala Ala Thr Glu Ala

180 185 190

Pro Ser Ser Thr Pro Lys Ala Gln Gly Pro Ala Ala Ser Ala Glu Glu

195 200 205

Pro Lys Pro Val Glu Ala Pro Ala Ala Asn Ser Asp Gln Thr Val Thr

210 215 220

Val Lys Glu

225

<210> 11

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 11

Lys Lys Lys Lys

<210> 12

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 12

Lys Lys Lys Lys Lys

1 5

<210> 13

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 13

Arg Arg Arg Arg

<210> 14

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 14

Arg Arg Arg Arg Arg

1 5

<210> 15

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<220>

<221> MOD_RES

<222> (1)..(4)

<223> X = Lys or Arg

<400> 15

Xaa Xaa Xaa Xaa

<210> 16

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<222> (1)..(5)

<223> Xaa can be any naturally occurring amino acid

<400> 16

Xaa Xaa Xaa Xaa Xaa

1 5

<210> 17

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 17

Gly Gly Lys Leu Ser Lys Lys

1 5

<210> 18

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 18

Gly Ala Lys Leu Ser Lys Lys

1 5

<210> 19

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 19

Gly Gly Lys Gln Ser Lys Lys

1 5

<210> 20

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 20

Gly Gly Lys Leu Ala Lys Lys

1 5

<210> 21

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 21

Gly Gly Lys Leu Ser Lys Lys

1 5

<210> 22

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 22

Gly Gly Lys Leu Ser Lys Lys Lys

1 5

<210> 23

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 23

Gly Gly Lys Leu Ser Lys Lys Ser

1 5

<210> 24

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 24

Gly Ala Lys Leu Ser Lys Lys Lys

1 5

<210> 25

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 25

Gly Ala Lys Leu Ser Lys Lys Ser

1 5

<210> 26

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 26

Gly Gly Lys Gln Ser Lys Lys Lys

1 5

<210> 27

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 27

Gly Gly Lys Gln Ser Lys Lys Ser

1 5

<210> 28

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 28

Gly Gly Lys Leu Ala Lys Lys Lys

1 5

<210> 29

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 29

Gly Gly Lys Leu Ala Lys Lys Ser

1 5

<210> 30

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 30

Gly Gly Gly Gly

<210> 31

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<222> (1)..(4)

<223> a linker; may contain 1 to 100 sequence repeats

<400> 31

Gly Gly Gly Ser

<210> 32

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 32

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn

1 5 10

<210> 33

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 33

Gly Ala Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn

1 5 10

<210> 34

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 34

Gly Gly Lys Gln Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn

1 5 10

<210> 35

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 35

Gly Gly Lys Leu Ala Lys Lys Lys Lys Gly Tyr Asn Val Asn

1 5 10

<210> 36

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 36

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Ser Gly Gly

1 5 10

<210> 37

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 37

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Ser Gly Gly Ser

1 5 10

<210> 38

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 38

Gly Gly Lys Leu Ser Lys Lys Lys Lys Ser Gly Gly Ser Gly

1 5 10

<210> 39

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 39

Gly Gly Lys Leu Ser Lys Lys Lys Ser Gly Gly Ser Gly Gly

1 5 10

<210> 40

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 40

Gly Gly Lys Leu Ser Lys Lys Ser Gly Gly Ser Gly Gly Ser

1 5 10

<210> 41

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 41

Gly Gly Lys Leu Ser Lys Ser Gly Gly Ser Gly Gly Ser Val

1 5 10

<210> 42

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 42

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys Ser

1 5 10

<210> 43

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 453 to 459 of SEQ44

<400> 43

Gly Thr Thr Thr Gln Ser Arg

1 5

<210> 44

<211> 735

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AAV2 VP1 (virus)

<400> 44

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr

580 585 590

Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp

595 600 605

Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr

610 615 620

Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys

625 630 635 640

His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn

645 650 655

Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln

660 665 670

Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys

675 680 685

Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr

690 695 700

Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr

705 710 715 720

Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 45

<211> 653

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> REP1

<400> 45

Met Ala Asp Thr Leu Pro Ser Glu Phe Asp Val Ile Val Ile Gly Thr

1 5 10 15

Gly Leu Pro Glu Ser Ile Ile Ala Ala Ala Cys Ser Arg Ser Gly Arg

20 25 30

Arg Val Leu His Val Asp Ser Arg Ser Tyr Tyr Gly Gly Asn Trp Ala

35 40 45

Ser Phe Ser Phe Ser Gly Leu Leu Ser Trp Leu Lys Glu Tyr Gln Glu

50 55 60

Asn Ser Asp Ile Val Ser Asp Ser Pro Val Trp Gln Asp Gln Ile Leu

65 70 75 80

Glu Asn Glu Glu Ala Ile Ala Leu Ser Arg Lys Asp Lys Thr Ile Gln

85 90 95

His Val Glu Val Phe Cys Tyr Ala Ser Gln Asp Leu His Glu Asp Val

100 105 110

Glu Glu Ala Gly Ala Leu Gln Lys Asn His Ala Leu Val Thr Ser Ala

115 120 125

Asn Ser Thr Glu Ala Ala Asp Ser Ala Phe Leu Pro Thr Glu Asp Glu

130 135 140

Ser Leu Ser Thr Met Ser Cys Glu Met Leu Thr Glu Gln Thr Pro Ser

145 150 155 160

Ser Asp Pro Glu Asn Ala Leu Glu Val Asn Gly Ala Glu Val Thr Gly

165 170 175

Glu Lys Glu Asn His Cys Asp Asp Lys Thr Cys Val Pro Ser Thr Ser

180 185 190

Ala Glu Asp Met Ser Glu Asn Val Pro Ile Ala Glu Asp Thr Thr Glu

195 200 205

Gln Pro Lys Lys Asn Arg Ile Thr Tyr Ser Gln Ile Ile Lys Glu Gly

210 215 220

Arg Arg Phe Asn Ile Asp Leu Val Ser Lys Leu Leu Tyr Ser Arg Gly

225 230 235 240

Leu Leu Ile Asp Leu Leu Ile Lys Ser Asn Val Ser Arg Tyr Ala Glu

245 250 255

Phe Lys Asn Ile Thr Arg Ile Leu Ala Phe Arg Glu Gly Arg Val Glu

260 265 270

Gln Val Pro Cys Ser Arg Ala Asp Val Phe Asn Ser Lys Gln Leu Thr

275 280 285

Met Val Glu Lys Arg Met Leu Met Lys Phe Leu Thr Phe Cys Met Glu

290 295 300

Tyr Glu Lys Tyr Pro Asp Glu Tyr Lys Gly Tyr Glu Glu Ile Thr Phe

305 310 315 320

Tyr Glu Tyr Leu Lys Thr Gln Lys Leu Thr Pro Asn Leu Gln Tyr Ile

325 330 335

Val Met His Ser Ile Ala Met Thr Ser Glu Thr Ala Ser Ser Thr Ile

340 345 350

Asp Gly Leu Lys Ala Thr Lys Asn Phe Leu His Cys Leu Gly Arg Tyr

355 360 365

Gly Asn Thr Pro Phe Leu Phe Pro Leu Tyr Gly Gln Gly Glu Leu Pro

370 375 380

Gln Cys Phe Cys Arg Met Cys Ala Val Phe Gly Gly Ile Tyr Cys Leu

385 390 395 400

Arg His Ser Val Gln Cys Leu Val Val Asp Lys Glu Ser Arg Lys Cys

405 410 415

Lys Ala Ile Ile Asp Gln Phe Gly Gln Arg Ile Ile Ser Glu His Phe

420 425 430

Leu Val Glu Asp Ser Tyr Phe Pro Glu Asn Met Cys Ser Arg Val Gln

435 440 445

Tyr Arg Gln Ile Ser Arg Ala Val Leu Ile Thr Asp Arg Ser Val Leu

450 455 460

Lys Thr Asp Ser Asp Gln Gln Ile Ser Ile Leu Thr Val Pro Ala Glu

465 470 475 480

Glu Pro Gly Thr Phe Ala Val Arg Val Ile Glu Leu Cys Ser Ser Thr

485 490 495

Met Thr Cys Met Lys Gly Thr Tyr Leu Val His Leu Thr Cys Thr Ser

500 505 510

Ser Lys Thr Ala Arg Glu Asp Leu Glu Ser Val Val Gln Lys Leu Phe

515 520 525

Val Pro Tyr Thr Glu Met Glu Ile Glu Asn Glu Gln Val Glu Lys Pro

530 535 540

Arg Ile Leu Trp Ala Leu Tyr Phe Asn Met Arg Asp Ser Ser Asp Ile

545 550 555 560

Ser Arg Ser Cys Tyr Asn Asp Leu Pro Ser Asn Val Tyr Val Cys Ser

565 570 575

Gly Pro Asp Cys Gly Leu Gly Asn Asp Asn Ala Val Lys Gln Ala Glu

580 585 590

Thr Leu Phe Gln Glu Ile Cys Pro Asn Glu Asp Phe Cys Pro Pro Pro

595 600 605

Pro Asn Pro Glu Asp Ile Ile Leu Asp Gly Asp Ser Leu Gln Pro Glu

610 615 620

Ala Ser Glu Ser Ser Ala Ile Pro Glu Ala Asn Ser Glu Thr Phe Lys

625 630 635 640

Glu Ser Thr Asn Leu Gly Asn Leu Glu Glu Ser Ser Glu

645 650

<210> 46

<211> 2

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(2)

<223> [ (Gly) n-Ser ] m shortest example of linker, where n is

Any integer from 1 to 100, and m is any integer from 1 to 100

<400> 46

Gly Ser

<210> 47

<211> 2

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(2)

<223> [ (Gly) x-Sery ] z linker, where x is

An integer from 1 to 4, y is 0 or 1, and z is from 1 to

An integer of 50

<400> 47

Gly Ser

<210> 48

<211> 1

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> shortest example of Gn linker, where n can be from

An integer of 1 to 100

<400> 48

Gly

<210> 49

<211> 2

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(2)

<223> (GlyAla) shortest example of n linker, where n is

An integer between 1 and 100

<400> 49

Gly Ala

<210> 50

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(3)

<223> a linker; may contain 1 to 100 sequence repeats

<400> 50

Gly Gly Ser

<210> 51

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 51

Gly Gly Lys Leu Ser Lys

1 5

<210> 52

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 52

Gly Ala Lys Leu Ser Lys

1 5

<210> 53

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 53

Gly Gly Lys Gln Ser Lys

1 5

<210> 54

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 54

Gly Gly Lys Leu Ala Lys

1 5

<210> 55

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 55

Lys Lys Lys Gly

<210> 56

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 56

Lys Lys Lys Gly Tyr

1 5

<210> 57

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 57

Lys Lys Lys Gly Tyr Asn

1 5

<210> 58

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 58

Lys Lys Lys Gly Tyr Asn Val

1 5

<210> 59

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 59

Lys Lys Lys Gly Tyr Asn Val Asn

1 5

<210> 60

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 60

Lys Lys Lys Gly Tyr Ser

1 5

<210> 61

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 61

Lys Lys Lys Gly Tyr Gly

1 5

<210> 62

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 62

Lys Lys Lys Gly Tyr Gly Gly

1 5

<210> 63

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 63

Lys Lys Lys Gly Ser

1 5

<210> 64

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 64

Lys Lys Lys Gly Ser Gly

1 5

<210> 65

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 65

Lys Lys Lys Gly Ser Gly

1 5

<210> 66

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 66

Lys Lys Lys Gly Ser Gly Ser

1 5

<210> 67

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 67

Lys Lys Lys Ser

<210> 68

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 68

Lys Lys Lys Ser Gly

1 5

<210> 69

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 69

Lys Lys Lys Ser Gly Gly

1 5

<210> 70

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 70

Lys Lys Lys Ser Gly Gly Ser

1 5

<210> 71

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 71

Lys Lys Lys Ser Gly Gly Ser Gly

1 5

<210> 72

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 72

Lys Lys Ser Gly Gly Ser Gly Gly

1 5

<210> 73

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 73

Lys Lys Lys Ser Gly Gly Ser Gly Gly Ser

1 5 10

<210> 74

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 74

Lys Arg Phe Ser Phe Lys Lys Ser

1 5

<210> 75

<211> 29

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 75

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp Glu

1 5 10 15

Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly Ala Ala

20 25

<210> 76

<211> 28

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 76

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp Glu

1 5 10 15

Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly Ala

20 25

<210> 77

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 77

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp Glu

1 5 10 15

Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu Gly

20 25

<210> 78

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 78

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp Glu

1 5 10 15

Lys Ala Lys Glu Lys Asp Lys Lys Ala Glu

20 25

<210> 79

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 79

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp Glu

1 5 10 15

Lys Ala Lys Glu Lys Asp Lys Lys Ala

20 25

<210> 80

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 80

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp Glu

1 5 10 15

Lys Ala Lys Glu Lys Asp Lys Lys

<210> 81

<211> 23

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 81

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp Glu

1 5 10 15

Lys Ala Lys Glu Lys Asp Lys

<210> 82

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 82

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp Glu

1 5 10 15

Lys Ala Lys Glu Lys Asp

<210> 83

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 83

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp Glu

1 5 10 15

Lys Ala Lys Glu Lys

<210> 84

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 84

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp Glu

1 5 10 15

Lys Ala Lys Glu

<210> 85

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 85

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp Glu

1 5 10 15

Lys Ala Lys

<210> 86

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 86

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp Glu

1 5 10 15

Lys Ala

<210> 87

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 87

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp Glu

1 5 10 15

Lys

<210> 88

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 88

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp Glu

1 5 10 15

<210> 89

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 89

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val Asn Asp

1 5 10 15

<210> 90

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 90

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn Val

1 5 10

<210> 91

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 91

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr Asn

1 5 10

<210> 92

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 92

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly Tyr

1 5 10

<210> 93

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 93

Gly Gly Lys Leu Ser Lys Lys Lys Lys Gly

1 5 10

<210> 94

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 94

Gly Gly Lys Leu Ser Lys Lys Lys Lys

1 5

<210> 95

<211> 29

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 95

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser Phe Lys Lys Asn Lys Lys Glu Ala

20 25

<210> 96

<211> 28

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 96

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser Phe Lys Lys Asn Lys Lys Glu

20 25

<210> 97

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 97

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser Phe Lys Lys Asn Lys Lys

20 25

<210> 98

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 98

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser Phe Lys Lys Asn Lys

20 25

<210> 99

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 99

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser Phe Lys Lys Asn

20 25

<210> 100

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 100

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser Phe Lys Lys

<210> 101

<211> 23

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 101

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser Phe Lys

<210> 102

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 102

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser Phe

<210> 103

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 103

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser

<210> 104

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 104

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe

<210> 105

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 105

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly

<210> 106

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 106

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser

<210> 107

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 107

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu

<210> 108

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 108

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

<210> 109

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 109

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe

1 5 10 15

<210> 110

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 110

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys Lys

1 5 10

<210> 111

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 111

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe Lys

1 5 10

<210> 112

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 112

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser Phe

1 5 10

<210> 113

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 113

Gly Ala Lys Lys Ser Lys Lys Arg Phe Ser

1 5 10

<210> 114

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 114

Gly Ala Lys Lys Ser Lys Lys Arg Phe

1 5

<210> 115

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 115

Gly Ala Lys Lys Ser Lys Lys Arg

1 5

<210> 116

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 116

Gly Ala Lys Lys Ser Lys Lys

1 5

<210> 117

<211> 29

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 117

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser Phe Lys Lys Asn Lys Lys Glu Ala

20 25

<210> 118

<211> 28

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 118

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser Phe Lys Lys Asn Lys Lys Glu

20 25

<210> 119

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 119

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser Phe Lys Lys Asn Lys Lys

20 25

<210> 120

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 120

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser Phe Lys Lys Asn Lys

20 25

<210> 121

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 121

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser Phe Lys Lys Asn

20 25

<210> 122

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 122

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser Phe Lys Lys

<210> 123

<211> 23

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 123

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser Phe Lys

<210> 124

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 124

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser Phe

<210> 125

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 125

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe Ser

<210> 126

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 126

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly Phe

<210> 127

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 127

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser Gly

<210> 128

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 128

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu Ser

<210> 129

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 129

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

Leu

<210> 130

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 130

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe Lys

1 5 10 15

<210> 131

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 131

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe

1 5 10 15

<210> 132

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 132

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys Ser

1 5 10

<210> 133

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 133

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys Lys

1 5 10

<210> 134

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 134

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe Lys

1 5 10

<210> 135

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 135

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser Phe

1 5 10

<210> 136

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 136

Gly Ala Lys Lys Ala Lys Lys Arg Phe Ser

1 5 10

<210> 137

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 137

Gly Ala Lys Lys Ala Lys Lys Arg Phe

1 5

<210> 138

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 138

Gly Ala Lys Lys Ala Lys Lys Arg

1 5

<210> 139

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 139

Gly Ala Lys Lys Ala Lys Lys

1 5

<210> 140

<211> 28

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 140

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys Arg Phe Ser Phe Lys

1 5 10 15

Lys Ser Phe Lys Leu Ser Gly Phe Ser Phe Lys Lys

20 25

<210> 141

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 141

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys Arg Phe Ser Phe Lys

1 5 10 15

Lys Ser Phe Lys Leu Ser Gly Phe Ser Phe Lys

20 25

<210> 142

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 142

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys Arg Phe Ser Phe Lys

1 5 10 15

Lys Ser Phe Lys Leu Ser Gly Phe Ser Phe

20 25

<210> 143

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 143

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys Arg Phe Ser Phe Lys

1 5 10 15

Lys Ser Phe Lys Leu Ser Gly Phe Ser

20 25

<210> 144

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 144

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys Arg Phe Ser Phe Lys

1 5 10 15

Lys Ser Phe Lys Leu Ser Gly Phe

<210> 145

<211> 23

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 145

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys Arg Phe Ser Phe Lys

1 5 10 15

Lys Ser Phe Lys Leu Ser Gly

<210> 146

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 146

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys Arg Phe Ser Phe Lys

1 5 10 15

Lys Ser Phe Lys Leu Ser

<210> 147

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 147

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys Arg Phe Ser Phe Lys

1 5 10 15

Lys Ser Phe Lys Leu

<210> 148

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 148

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys Arg Phe Ser Phe Lys

1 5 10 15

Lys Ser Phe Lys

<210> 149

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 149

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys Arg Phe Ser Phe Lys

1 5 10 15

Lys Ser Phe

<210> 150

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 150

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys Arg Phe Ser Phe Lys

1 5 10 15

Lys Ser

<210> 151

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 151

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys Arg Phe Ser Phe Lys

1 5 10 15

Lys

<210> 152

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 152

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys Arg Phe Ser Phe Lys

1 5 10 15

<210> 153

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 153

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys Arg Phe Ser Phe

1 5 10 15

<210> 154

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 154

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys Arg Phe Ser

1 5 10

<210> 155

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 155

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys Arg Phe

1 5 10

<210> 156

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 156

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys Arg

1 5 10

<210> 157

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 157

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys Lys

1 5 10

<210> 158

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 158

Gly Ala Gln Glu Ser Lys Lys Lys Lys Lys

1 5 10

<210> 159

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 159

Gly Ala Gln Glu Ser Lys Lys Lys Lys

1 5

<210> 160

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 160

Gly Ala Gln Glu Ser Lys Lys Lys

1 5

<210> 161

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 161

Gly Ala Gln Glu Ser Lys Lys

1 5

<210> 162

<211> 30

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 162

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys

1 5 10 15

Pro Phe Lys Leu Ser Gly Leu Ser Phe Lys Arg Asn Arg Lys

20 25 30

<210> 163

<211> 29

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 163

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys

1 5 10 15

Pro Phe Lys Leu Ser Gly Leu Ser Phe Lys Arg Asn Arg

20 25

<210> 164

<211> 28

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 164

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys

1 5 10 15

Pro Phe Lys Leu Ser Gly Leu Ser Phe Lys Arg Asn

20 25

<210> 165

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 165

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys

1 5 10 15

Pro Phe Lys Leu Ser Gly Leu Ser Phe Lys Arg

20 25

<210> 166

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 166

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys

1 5 10 15

Pro Phe Lys Leu Ser Gly Leu Ser Phe Lys

20 25

<210> 167

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 167

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys

1 5 10 15

Pro Phe Lys Leu Ser Gly Leu Ser Phe

20 25

<210> 168

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 168

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys

1 5 10 15

Pro Phe Lys Leu Ser Gly Leu Ser

<210> 169

<211> 23

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 169

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys

1 5 10 15

Pro Phe Lys Leu Ser Gly Leu

<210> 170

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 170

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys

1 5 10 15

Pro Phe Lys Leu Ser Gly

<210> 171

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 171

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys

1 5 10 15

Pro Phe Lys Leu Ser

<210> 172

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 172

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys

1 5 10 15

Pro Phe Lys Leu

<210> 173

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 173

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys

1 5 10 15

Pro Phe Lys

<210> 174

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 174

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys

1 5 10 15

Pro Phe

<210> 175

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 175

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys

1 5 10 15

Pro

<210> 176

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 176

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser Phe Lys Lys

1 5 10 15

<210> 177

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 177

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser Phe Lys

1 5 10 15

<210> 178

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 178

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser Phe

1 5 10

<210> 179

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 179

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe Ser

1 5 10

<210> 180

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 180

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys Phe

1 5 10

<210> 181

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 181

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys Lys

1 5 10

<210> 182

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 182

Gly Ser Gln Ser Ser Lys Lys Lys Lys Lys

1 5 10

<210> 183

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 183

Gly Ser Gln Ser Ser Lys Lys Lys Lys

1 5

<210> 184

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 184

Gly Ser Gln Ser Ser Lys Lys Lys

1 5

<210> 185

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 185

Gly Ser Gln Ser Ser Lys Lys

1 5

<210> 186

<211> 731

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 186

Pro Ser Ala Arg Pro Pro Pro Ser Leu Ser Leu Arg Glu Gly Glu Pro

1 5 10 15

Phe Glu Leu Arg Cys Thr Ala Ala Ser Ala Ser Pro Leu His Thr His

20 25 30

Leu Ala Leu Leu Trp Glu Val His Arg Gly Pro Ala Arg Arg Ser Val

35 40 45

Leu Ala Leu Thr His Glu Gly Arg Phe His Pro Gly Leu Gly Tyr Glu

50 55 60

Gln Arg Tyr His Ser Gly Asp Val Arg Leu Asp Thr Val Gly Ser Asp

65 70 75 80

Ala Tyr Arg Leu Ser Val Ser Arg Ala Leu Ser Ala Asp Gln Gly Ser

85 90 95

Tyr Arg Cys Ile Val Ser Glu Trp Ile Ala Glu Gln Gly Asn Trp Gln

100 105 110

Glu Ile Gln Glu Lys Ala Val Glu Val Ala Thr Val Val Ile Gln Pro

115 120 125

Ser Val Leu Arg Ala Ala Val Pro Lys Asn Val Ser Val Ala Glu Gly

130 135 140

Lys Glu Leu Asp Leu Thr Cys Asn Ile Thr Thr Asp Arg Ala Asp Asp

145 150 155 160

Val Arg Pro Glu Val Thr Trp Ser Phe Ser Arg Met Pro Asp Ser Thr

165 170 175

Leu Pro Gly Ser Arg Val Leu Ala Arg Leu Asp Arg Asp Ser Leu Val

180 185 190

His Ser Ser Pro His Val Ala Leu Ser His Val Asp Ala Arg Ser Tyr

195 200 205

His Leu Leu Val Arg Asp Val Ser Lys Glu Asn Ser Gly Tyr Tyr Tyr

210 215 220

Cys His Val Ser Leu Trp Ala Pro Gly His Asn Arg Ser Trp His Lys

225 230 235 240

Val Ala Glu Ala Val Ser Ser Pro Ala Gly Val Gly Val Thr Trp Leu

245 250 255

Glu Pro Asp Tyr Gln Val Tyr Leu Asn Ala Ser Lys Val Pro Gly Phe

260 265 270

Ala Asp Asp Pro Thr Glu Leu Ala Cys Arg Val Val Asp Thr Lys Ser

275 280 285

Gly Glu Ala Asn Val Arg Phe Thr Val Ser Trp Tyr Tyr Arg Met Asn

290 295 300

Arg Arg Ser Asp Asn Val Val Thr Ser Glu Leu Leu Ala Val Met Asp

305 310 315 320

Gly Asp Trp Thr Leu Lys Tyr Gly Glu Arg Ser Lys Gln Arg Ala Gln

325 330 335

Asp Gly Asp Phe Ile Phe Ser Lys Glu His Thr Asp Thr Phe Asn Phe

340 345 350

Arg Ile Gln Arg Thr Thr Glu Glu Asp Arg Gly Asn Tyr Tyr Cys Val

355 360 365

Val Ser Ala Trp Thr Lys Gln Arg Asn Asn Ser Trp Val Lys Ser Lys

370 375 380

Asp Val Phe Ser Lys Pro Val Asn Ile Phe Trp Ala Leu Glu Asp Ser

385 390 395 400

Val Leu Val Val Lys Ala Arg Gln Pro Lys Pro Phe Phe Ala Ala Gly

405 410 415

Asn Thr Phe Glu Met Thr Cys Lys Val Ser Ser Lys Asn Ile Lys Ser

420 425 430

Pro Arg Tyr Ser Val Leu Ile Met Ala Glu Lys Pro Val Gly Asp Leu

435 440 445

Ser Ser Pro Asn Glu Thr Lys Tyr Ile Ile Ser Leu Asp Gln Asp Ser

450 455 460

Val Val Lys Leu Glu Asn Trp Thr Asp Ala Ser Arg Val Asp Gly Val

465 470 475 480

Val Leu Glu Lys Val Gln Glu Asp Glu Phe Arg Tyr Arg Met Tyr Gln

485 490 495

Thr Gln Val Ser Asp Ala Gly Leu Tyr Arg Cys Met Val Thr Ala Trp

500 505 510

Ser Pro Val Arg Gly Ser Leu Trp Arg Glu Ala Ala Thr Ser Leu Ser

515 520 525

Asn Pro Ile Glu Ile Asp Phe Gln Thr Ser Gly Pro Ile Phe Asn Ala

530 535 540

Ser Val His Ser Asp Thr Pro Ser Val Ile Arg Gly Asp Leu Ile Lys

545 550 555 560

Leu Phe Cys Ile Ile Thr Val Glu Gly Ala Ala Leu Asp Pro Asp Asp

565 570 575

Met Ala Phe Asp Val Ser Trp Phe Ala Val His Ser Phe Gly Leu Asp

580 585 590

Lys Ala Pro Val Leu Leu Ser Ser Leu Asp Arg Lys Gly Ile Val Thr

595 600 605

Thr Ser Arg Arg Asp Trp Lys Ser Asp Leu Ser Leu Glu Arg Val Ser

610 615 620

Val Leu Glu Phe Leu Leu Gln Val His Gly Ser Glu Asp Gln Asp Phe

625 630 635 640

Gly Asn Tyr Tyr Cys Ser Val Thr Pro Trp Val Lys Ser Pro Thr Gly

645 650 655

Ser Trp Gln Lys Glu Ala Glu Ile His Ser Lys Pro Val Phe Ile Thr

660 665 670

Val Lys Met Asp Val Leu Asn Ala Phe Lys Tyr Pro Leu Leu Ile Gly

675 680 685

Val Gly Leu Ser Thr Val Ile Gly Leu Leu Ser Cys Leu Ile Gly Tyr

690 695 700

Cys Ser Ser His Trp Cys Cys Lys Lys Glu Val Gln Glu Thr Arg Arg

705 710 715 720

Glu Arg Arg Arg Leu Met Ser Met Glu Met Asp

725 730

<210> 187

<211> 611

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 187

Val Ala Thr Val Val Ile Gln Pro Ser Val Leu Arg Ala Ala Val Pro

1 5 10 15

Lys Asn Val Ser Val Ala Glu Gly Lys Glu Leu Asp Leu Thr Cys Asn

20 25 30

Ile Thr Thr Asp Arg Ala Asp Asp Val Arg Pro Glu Val Thr Trp Ser

35 40 45

Phe Ser Arg Met Pro Asp Ser Thr Leu Pro Gly Ser Arg Val Leu Ala

50 55 60

Arg Leu Asp Arg Asp Ser Leu Val His Ser Ser Pro His Val Ala Leu

65 70 75 80

Ser His Val Asp Ala Arg Ser Tyr His Leu Leu Val Arg Asp Val Ser

85 90 95

Lys Glu Asn Ser Gly Tyr Tyr Tyr Cys His Val Ser Leu Trp Ala Pro

100 105 110

Gly His Asn Arg Ser Trp His Lys Val Ala Glu Ala Val Ser Ser Pro

115 120 125

Ala Gly Val Gly Val Thr Trp Leu Glu Pro Asp Tyr Gln Val Tyr Leu

130 135 140

Asn Ala Ser Lys Val Pro Gly Phe Ala Asp Asp Pro Thr Glu Leu Ala

145 150 155 160

Cys Arg Val Val Asp Thr Lys Ser Gly Glu Ala Asn Val Arg Phe Thr

165 170 175

Val Ser Trp Tyr Tyr Arg Met Asn Arg Arg Ser Asp Asn Val Val Thr

180 185 190

Ser Glu Leu Leu Ala Val Met Asp Gly Asp Trp Thr Leu Lys Tyr Gly

195 200 205

Glu Arg Ser Lys Gln Arg Ala Gln Asp Gly Asp Phe Ile Phe Ser Lys

210 215 220

Glu His Thr Asp Thr Phe Asn Phe Arg Ile Gln Arg Thr Thr Glu Glu

225 230 235 240

Asp Arg Gly Asn Tyr Tyr Cys Val Val Ser Ala Trp Thr Lys Gln Arg

245 250 255

Asn Asn Ser Trp Val Lys Ser Lys Asp Val Phe Ser Lys Pro Val Asn

260 265 270

Ile Phe Trp Ala Leu Glu Asp Ser Val Leu Val Val Lys Ala Arg Gln

275 280 285

Pro Lys Pro Phe Phe Ala Ala Gly Asn Thr Phe Glu Met Thr Cys Lys

290 295 300

Val Ser Ser Lys Asn Ile Lys Ser Pro Arg Tyr Ser Val Leu Ile Met

305 310 315 320

Ala Glu Lys Pro Val Gly Asp Leu Ser Ser Pro Asn Glu Thr Lys Tyr

325 330 335

Ile Ile Ser Leu Asp Gln Asp Ser Val Val Lys Leu Glu Asn Trp Thr

340 345 350

Asp Ala Ser Arg Val Asp Gly Val Val Leu Glu Lys Val Gln Glu Asp

355 360 365

Glu Phe Arg Tyr Arg Met Tyr Gln Thr Gln Val Ser Asp Ala Gly Leu

370 375 380

Tyr Arg Cys Met Val Thr Ala Trp Ser Pro Val Arg Gly Ser Leu Trp

385 390 395 400

Arg Glu Ala Ala Thr Ser Leu Ser Asn Pro Ile Glu Ile Asp Phe Gln

405 410 415

Thr Ser Gly Pro Ile Phe Asn Ala Ser Val His Ser Asp Thr Pro Ser

420 425 430

Val Ile Arg Gly Asp Leu Ile Lys Leu Phe Cys Ile Ile Thr Val Glu

435 440 445

Gly Ala Ala Leu Asp Pro Asp Asp Met Ala Phe Asp Val Ser Trp Phe

450 455 460

Ala Val His Ser Phe Gly Leu Asp Lys Ala Pro Val Leu Leu Ser Ser

465 470 475 480

Leu Asp Arg Lys Gly Ile Val Thr Thr Ser Arg Arg Asp Trp Lys Ser

485 490 495

Asp Leu Ser Leu Glu Arg Val Ser Val Leu Glu Phe Leu Leu Gln Val

500 505 510

His Gly Ser Glu Asp Gln Asp Phe Gly Asn Tyr Tyr Cys Ser Val Thr

515 520 525

Pro Trp Val Lys Ser Pro Thr Gly Ser Trp Gln Lys Glu Ala Glu Ile

530 535 540

His Ser Lys Pro Val Phe Ile Thr Val Lys Met Asp Val Leu Asn Ala

545 550 555 560

Phe Lys Tyr Pro Leu Leu Ile Gly Val Gly Leu Ser Thr Val Ile Gly

565 570 575

Leu Leu Ser Cys Leu Ile Gly Tyr Cys Ser Ser His Trp Cys Cys Lys

580 585 590

Lys Glu Val Gln Glu Thr Arg Arg Glu Arg Arg Arg Leu Met Ser Met

595 600 605

Glu Met Asp

610

<210> 188

<211> 485

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 188

Ser Pro Ala Gly Val Gly Val Thr Trp Leu Glu Pro Asp Tyr Gln Val

1 5 10 15

Tyr Leu Asn Ala Ser Lys Val Pro Gly Phe Ala Asp Asp Pro Thr Glu

20 25 30

Leu Ala Cys Arg Val Val Asp Thr Lys Ser Gly Glu Ala Asn Val Arg

35 40 45

Phe Thr Val Ser Trp Tyr Tyr Arg Met Asn Arg Arg Ser Asp Asn Val

50 55 60

Val Thr Ser Glu Leu Leu Ala Val Met Asp Gly Asp Trp Thr Leu Lys

65 70 75 80

Tyr Gly Glu Arg Ser Lys Gln Arg Ala Gln Asp Gly Asp Phe Ile Phe

85 90 95

Ser Lys Glu His Thr Asp Thr Phe Asn Phe Arg Ile Gln Arg Thr Thr

100 105 110

Glu Glu Asp Arg Gly Asn Tyr Tyr Cys Val Val Ser Ala Trp Thr Lys

115 120 125

Gln Arg Asn Asn Ser Trp Val Lys Ser Lys Asp Val Phe Ser Lys Pro

130 135 140

Val Asn Ile Phe Trp Ala Leu Glu Asp Ser Val Leu Val Val Lys Ala

145 150 155 160

Arg Gln Pro Lys Pro Phe Phe Ala Ala Gly Asn Thr Phe Glu Met Thr

165 170 175

Cys Lys Val Ser Ser Lys Asn Ile Lys Ser Pro Arg Tyr Ser Val Leu

180 185 190

Ile Met Ala Glu Lys Pro Val Gly Asp Leu Ser Ser Pro Asn Glu Thr

195 200 205

Lys Tyr Ile Ile Ser Leu Asp Gln Asp Ser Val Val Lys Leu Glu Asn

210 215 220

Trp Thr Asp Ala Ser Arg Val Asp Gly Val Val Leu Glu Lys Val Gln

225 230 235 240

Glu Asp Glu Phe Arg Tyr Arg Met Tyr Gln Thr Gln Val Ser Asp Ala

245 250 255

Gly Leu Tyr Arg Cys Met Val Thr Ala Trp Ser Pro Val Arg Gly Ser

260 265 270

Leu Trp Arg Glu Ala Ala Thr Ser Leu Ser Asn Pro Ile Glu Ile Asp

275 280 285

Phe Gln Thr Ser Gly Pro Ile Phe Asn Ala Ser Val His Ser Asp Thr

290 295 300

Pro Ser Val Ile Arg Gly Asp Leu Ile Lys Leu Phe Cys Ile Ile Thr

305 310 315 320

Val Glu Gly Ala Ala Leu Asp Pro Asp Asp Met Ala Phe Asp Val Ser

325 330 335

Trp Phe Ala Val His Ser Phe Gly Leu Asp Lys Ala Pro Val Leu Leu

340 345 350

Ser Ser Leu Asp Arg Lys Gly Ile Val Thr Thr Ser Arg Arg Asp Trp

355 360 365

Lys Ser Asp Leu Ser Leu Glu Arg Val Ser Val Leu Glu Phe Leu Leu

370 375 380

Gln Val His Gly Ser Glu Asp Gln Asp Phe Gly Asn Tyr Tyr Cys Ser

385 390 395 400

Val Thr Pro Trp Val Lys Ser Pro Thr Gly Ser Trp Gln Lys Glu Ala

405 410 415

Glu Ile His Ser Lys Pro Val Phe Ile Thr Val Lys Met Asp Val Leu

420 425 430

Asn Ala Phe Lys Tyr Pro Leu Leu Ile Gly Val Gly Leu Ser Thr Val

435 440 445

Ile Gly Leu Leu Ser Cys Leu Ile Gly Tyr Cys Ser Ser His Trp Cys

450 455 460

Cys Lys Lys Glu Val Gln Glu Thr Arg Arg Glu Arg Arg Arg Leu Met

465 470 475 480

Ser Met Glu Met Asp

485

<210> 189

<211> 343

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 189

Lys Pro Val Asn Ile Phe Trp Ala Leu Glu Asp Ser Val Leu Val Val

1 5 10 15

Lys Ala Arg Gln Pro Lys Pro Phe Phe Ala Ala Gly Asn Thr Phe Glu

20 25 30

Met Thr Cys Lys Val Ser Ser Lys Asn Ile Lys Ser Pro Arg Tyr Ser

35 40 45

Val Leu Ile Met Ala Glu Lys Pro Val Gly Asp Leu Ser Ser Pro Asn

50 55 60

Glu Thr Lys Tyr Ile Ile Ser Leu Asp Gln Asp Ser Val Val Lys Leu

65 70 75 80

Glu Asn Trp Thr Asp Ala Ser Arg Val Asp Gly Val Val Leu Glu Lys

85 90 95

Val Gln Glu Asp Glu Phe Arg Tyr Arg Met Tyr Gln Thr Gln Val Ser

100 105 110

Asp Ala Gly Leu Tyr Arg Cys Met Val Thr Ala Trp Ser Pro Val Arg

115 120 125

Gly Ser Leu Trp Arg Glu Ala Ala Thr Ser Leu Ser Asn Pro Ile Glu

130 135 140

Ile Asp Phe Gln Thr Ser Gly Pro Ile Phe Asn Ala Ser Val His Ser

145 150 155 160

Asp Thr Pro Ser Val Ile Arg Gly Asp Leu Ile Lys Leu Phe Cys Ile

165 170 175

Ile Thr Val Glu Gly Ala Ala Leu Asp Pro Asp Asp Met Ala Phe Asp

180 185 190

Val Ser Trp Phe Ala Val His Ser Phe Gly Leu Asp Lys Ala Pro Val

195 200 205

Leu Leu Ser Ser Leu Asp Arg Lys Gly Ile Val Thr Thr Ser Arg Arg

210 215 220

Asp Trp Lys Ser Asp Leu Ser Leu Glu Arg Val Ser Val Leu Glu Phe

225 230 235 240

Leu Leu Gln Val His Gly Ser Glu Asp Gln Asp Phe Gly Asn Tyr Tyr

245 250 255

Cys Ser Val Thr Pro Trp Val Lys Ser Pro Thr Gly Ser Trp Gln Lys

260 265 270

Glu Ala Glu Ile His Ser Lys Pro Val Phe Ile Thr Val Lys Met Asp

275 280 285

Val Leu Asn Ala Phe Lys Tyr Pro Leu Leu Ile Gly Val Gly Leu Ser

290 295 300

Thr Val Ile Gly Leu Leu Ser Cys Leu Ile Gly Tyr Cys Ser Ser His

305 310 315 320

Trp Cys Cys Lys Lys Glu Val Gln Glu Thr Arg Arg Glu Arg Arg Arg

325 330 335

Leu Met Ser Met Glu Met Asp

340

<210> 190

<211> 217

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 190

Val Arg Gly Ser Leu Trp Arg Glu Ala Ala Thr Ser Leu Ser Asn Pro

1 5 10 15

Ile Glu Ile Asp Phe Gln Thr Ser Gly Pro Ile Phe Asn Ala Ser Val

20 25 30

His Ser Asp Thr Pro Ser Val Ile Arg Gly Asp Leu Ile Lys Leu Phe

35 40 45

Cys Ile Ile Thr Val Glu Gly Ala Ala Leu Asp Pro Asp Asp Met Ala

50 55 60

Phe Asp Val Ser Trp Phe Ala Val His Ser Phe Gly Leu Asp Lys Ala

65 70 75 80

Pro Val Leu Leu Ser Ser Leu Asp Arg Lys Gly Ile Val Thr Thr Ser

85 90 95

Arg Arg Asp Trp Lys Ser Asp Leu Ser Leu Glu Arg Val Ser Val Leu

100 105 110

Glu Phe Leu Leu Gln Val His Gly Ser Glu Asp Gln Asp Phe Gly Asn

115 120 125

Tyr Tyr Cys Ser Val Thr Pro Trp Val Lys Ser Pro Thr Gly Ser Trp

130 135 140

Gln Lys Glu Ala Glu Ile His Ser Lys Pro Val Phe Ile Thr Val Lys

145 150 155 160

Met Asp Val Leu Asn Ala Phe Lys Tyr Pro Leu Leu Ile Gly Val Gly

165 170 175

Leu Ser Thr Val Ile Gly Leu Leu Ser Cys Leu Ile Gly Tyr Cys Ser

180 185 190

Ser His Trp Cys Cys Lys Lys Glu Val Gln Glu Thr Arg Arg Glu Arg

195 200 205

Arg Arg Leu Met Ser Met Glu Met Asp

210 215

<210> 191

<211> 66

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 191

Ser Lys Pro Val Phe Ile Thr Val Lys Met Asp Val Leu Asn Ala Phe

1 5 10 15

Lys Tyr Pro Leu Leu Ile Gly Val Gly Leu Ser Thr Val Ile Gly Leu

20 25 30

Leu Ser Cys Leu Ile Gly Tyr Cys Ser Ser His Trp Cys Cys Lys Lys

35 40 45

Glu Val Gln Glu Thr Arg Arg Glu Arg Arg Arg Leu Met Ser Met Glu

50 55 60

Met Asp

<210> 192

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 192

Met Gly Arg Leu Ala Ser Arg Pro Leu Leu Leu Ala Leu Leu Ser Leu

1 5 10 15

Ala Leu Cys Arg Gly

<210> 193

<211> 247

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 193

Pro Gly Thr Val Phe Thr Thr Val Glu Asp Leu Gly Ser Lys Ile Leu

1 5 10 15

Leu Thr Cys Ser Leu Asn Asp Ser Ala Thr Glu Val Thr Gly His Arg

20 25 30

Trp Leu Lys Gly Gly Val Val Leu Lys Glu Asp Ala Leu Pro Gly Gln

35 40 45

Lys Thr Glu Phe Lys Val Asp Ser Asp Asp Gln Trp Gly Glu Tyr Ser

50 55 60

Cys Val Phe Leu Pro Glu Pro Met Gly Thr Ala Asn Ile Gln Leu His

65 70 75 80

Gly Pro Pro Arg Val Lys Ala Val Lys Ser Ser Glu His Ile Asn Glu

85 90 95

Gly Glu Thr Ala Met Leu Val Cys Lys Ser Glu Ser Val Pro Pro Val

100 105 110

Thr Asp Trp Ala Trp Tyr Lys Ile Thr Asp Ser Glu Asp Lys Ala Leu

115 120 125

Met Asn Gly Ser Glu Ser Arg Phe Phe Val Ser Ser Ser Gln Gly Arg

130 135 140

Ser Glu Leu His Ile Glu Asn Leu Asn Met Glu Ala Asp Pro Gly Gln

145 150 155 160

Tyr Arg Cys Asn Gly Thr Ser Ser Lys Gly Ser Asp Gln Ala Ile Ile

165 170 175

Thr Leu Arg Val Arg Ser His Leu Ala Ala Leu Trp Pro Phe Leu Gly

180 185 190

Ile Val Ala Glu Val Leu Val Leu Val Thr Ile Ile Phe Ile Tyr Glu

195 200 205

Lys Arg Arg Lys Pro Glu Asp Val Leu Asp Asp Asp Asp Ala Gly Ser

210 215 220

Ala Pro Leu Lys Ser Ser Gly Gln His Gln Asn Asp Lys Gly Lys Asn

225 230 235 240

Val Arg Gln Arg Asn Ser Ser

245

<210> 194

<211> 168

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 194

His Gly Pro Pro Arg Val Lys Ala Val Lys Ser Ser Glu His Ile Asn

1 5 10 15

Glu Gly Glu Thr Ala Met Leu Val Cys Lys Ser Glu Ser Val Pro Pro

20 25 30

Val Thr Asp Trp Ala Trp Tyr Lys Ile Thr Asp Ser Glu Asp Lys Ala

35 40 45

Leu Met Asn Gly Ser Glu Ser Arg Phe Phe Val Ser Ser Ser Gln Gly

50 55 60

Arg Ser Glu Leu His Ile Glu Asn Leu Asn Met Glu Ala Asp Pro Gly

65 70 75 80

Gln Tyr Arg Cys Asn Gly Thr Ser Ser Lys Gly Ser Asp Gln Ala Ile

85 90 95

Ile Thr Leu Arg Val Arg Ser His Leu Ala Ala Leu Trp Pro Phe Leu

100 105 110

Gly Ile Val Ala Glu Val Leu Val Leu Val Thr Ile Ile Phe Ile Tyr

115 120 125

Glu Lys Arg Arg Lys Pro Glu Asp Val Leu Asp Asp Asp Asp Ala Gly

130 135 140

Ser Ala Pro Leu Lys Ser Ser Gly Gln His Gln Asn Asp Lys Gly Lys

145 150 155 160

Asn Val Arg Gln Arg Asn Ser Ser

165

<210> 195

<211> 66

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 195

Ser His Leu Ala Ala Leu Trp Pro Phe Leu Gly Ile Val Ala Glu Val

1 5 10 15

Leu Val Leu Val Thr Ile Ile Phe Ile Tyr Glu Lys Arg Arg Lys Pro

20 25 30

Glu Asp Val Leu Asp Asp Asp Asp Ala Gly Ser Ala Pro Leu Lys Ser

35 40 45

Ser Gly Gln His Gln Asn Asp Lys Gly Lys Asn Val Arg Gln Arg Asn

50 55 60

Ser Ser

<210> 196

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 196

Met Ala Ala Ala Leu Phe Val Leu Leu Gly Phe Ala Leu Leu Gly Thr

1 5 10 15

His Gly

<210> 197

<211> 456

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 197

Ala Pro Pro Gly Pro Arg Gly Arg Gln Ala Pro Thr Ser Pro Pro Arg

1 5 10 15

Met Thr Val His Glu Gly Gln Glu Leu Ala Leu Gly Cys Leu Ala Arg

20 25 30

Thr Ser Thr Gln Lys His Thr His Leu Ala Val Ser Phe Gly Arg Ser

35 40 45

Val Pro Glu Ala Pro Val Gly Arg Ser Thr Leu Gln Glu Val Val Gly

50 55 60

Ile Arg Ser Asp Leu Ala Val Glu Ala Gly Ala Pro Tyr Ala Glu Arg

65 70 75 80

Leu Ala Ala Gly Glu Leu Arg Leu Gly Lys Glu Gly Thr Asp Arg Tyr

85 90 95

Arg Met Val Val Gly Gly Ala Gln Ala Gly Asp Ala Gly Thr Tyr His

100 105 110

Cys Thr Ala Ala Glu Trp Ile Gln Asp Pro Asp Gly Ser Trp Ala Gln

115 120 125

Ile Ala Glu Lys Arg Ala Val Leu Ala His Val Asp Val Gln Thr Leu

130 135 140

Ser Ser Gln Leu Ala Val Thr Val Gly Pro Gly Glu Arg Arg Ile Gly

145 150 155 160

Pro Gly Glu Pro Leu Glu Leu Leu Cys Asn Val Ser Gly Ala Leu Pro

165 170 175

Pro Ala Gly Arg His Ala Ala Tyr Ser Val Gly Trp Glu Met Ala Pro

180 185 190

Ala Gly Ala Pro Gly Pro Gly Arg Leu Val Ala Gln Leu Asp Thr Glu

195 200 205

Gly Val Gly Ser Leu Gly Pro Gly Tyr Glu Gly Arg His Ile Ala Met

210 215 220

Glu Lys Val Ala Ser Arg Thr Tyr Arg Leu Arg Leu Glu Ala Ala Arg

225 230 235 240

Pro Gly Asp Ala Gly Thr Tyr Arg Cys Leu Ala Lys Ala Tyr Val Arg

245 250 255

Gly Ser Gly Thr Arg Leu Arg Glu Ala Ala Ser Ala Arg Ser Arg Pro

260 265 270

Leu Pro Val His Val Arg Glu Glu Gly Val Val Leu Glu Ala Val Ala

275 280 285

Trp Leu Ala Gly Gly Thr Val Tyr Arg Gly Glu Thr Ala Ser Leu Leu

290 295 300

Cys Asn Ile Ser Val Arg Gly Gly Pro Pro Gly Leu Arg Leu Ala Ala

305 310 315 320

Ser Trp Trp Val Glu Arg Pro Glu Asp Gly Glu Leu Ser Ser Val Pro

325 330 335

Ala Gln Leu Val Gly Gly Val Gly Gln Asp Gly Val Ala Glu Leu Gly

340 345 350

Val Arg Pro Gly Gly Gly Pro Val Ser Val Glu Leu Val Gly Pro Arg

355 360 365

Ser His Arg Leu Arg Leu His Ser Leu Gly Pro Glu Asp Glu Gly Val

370 375 380

Tyr His Cys Ala Pro Ser Ala Trp Val Gln His Ala Asp Tyr Ser Trp

385 390 395 400

Tyr Gln Ala Gly Ser Ala Arg Ser Gly Pro Val Thr Val Tyr Pro Tyr

405 410 415

Met His Ala Leu Asp Thr Leu Phe Val Pro Leu Leu Val Gly Thr Gly

420 425 430

Val Ala Leu Val Thr Gly Ala Thr Val Leu Gly Thr Ile Thr Cys Cys

435 440 445

Phe Met Lys Arg Leu Arg Lys Arg

450 455

<210> 198

<211> 320

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 198

Ala His Val Asp Val Gln Thr Leu Ser Ser Gln Leu Ala Val Thr Val

1 5 10 15

Gly Pro Gly Glu Arg Arg Ile Gly Pro Gly Glu Pro Leu Glu Leu Leu

20 25 30

Cys Asn Val Ser Gly Ala Leu Pro Pro Ala Gly Arg His Ala Ala Tyr

35 40 45

Ser Val Gly Trp Glu Met Ala Pro Ala Gly Ala Pro Gly Pro Gly Arg

50 55 60

Leu Val Ala Gln Leu Asp Thr Glu Gly Val Gly Ser Leu Gly Pro Gly

65 70 75 80

Tyr Glu Gly Arg His Ile Ala Met Glu Lys Val Ala Ser Arg Thr Tyr

85 90 95

Arg Leu Arg Leu Glu Ala Ala Arg Pro Gly Asp Ala Gly Thr Tyr Arg

100 105 110

Cys Leu Ala Lys Ala Tyr Val Arg Gly Ser Gly Thr Arg Leu Arg Glu

115 120 125

Ala Ala Ser Ala Arg Ser Arg Pro Leu Pro Val His Val Arg Glu Glu

130 135 140

Gly Val Val Leu Glu Ala Val Ala Trp Leu Ala Gly Gly Thr Val Tyr

145 150 155 160

Arg Gly Glu Thr Ala Ser Leu Leu Cys Asn Ile Ser Val Arg Gly Gly

165 170 175

Pro Pro Gly Leu Arg Leu Ala Ala Ser Trp Trp Val Glu Arg Pro Glu

180 185 190

Asp Gly Glu Leu Ser Ser Val Pro Ala Gln Leu Val Gly Gly Val Gly

195 200 205

Gln Asp Gly Val Ala Glu Leu Gly Val Arg Pro Gly Gly Gly Pro Val

210 215 220

Ser Val Glu Leu Val Gly Pro Arg Ser His Arg Leu Arg Leu His Ser

225 230 235 240

Leu Gly Pro Glu Asp Glu Gly Val Tyr His Cys Ala Pro Ser Ala Trp

245 250 255

Val Gln His Ala Asp Tyr Ser Trp Tyr Gln Ala Gly Ser Ala Arg Ser

260 265 270

Gly Pro Val Thr Val Tyr Pro Tyr Met His Ala Leu Asp Thr Leu Phe

275 280 285

Val Pro Leu Leu Val Gly Thr Gly Val Ala Leu Val Thr Gly Ala Thr

290 295 300

Val Leu Gly Thr Ile Thr Cys Cys Phe Met Lys Arg Leu Arg Lys Arg

305 310 315 320

<210> 199

<211> 179

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 199

Arg Glu Glu Gly Val Val Leu Glu Ala Val Ala Trp Leu Ala Gly Gly

1 5 10 15

Thr Val Tyr Arg Gly Glu Thr Ala Ser Leu Leu Cys Asn Ile Ser Val

20 25 30

Arg Gly Gly Pro Pro Gly Leu Arg Leu Ala Ala Ser Trp Trp Val Glu

35 40 45

Arg Pro Glu Asp Gly Glu Leu Ser Ser Val Pro Ala Gln Leu Val Gly

50 55 60

Gly Val Gly Gln Asp Gly Val Ala Glu Leu Gly Val Arg Pro Gly Gly

65 70 75 80

Gly Pro Val Ser Val Glu Leu Val Gly Pro Arg Ser His Arg Leu Arg

85 90 95

Leu His Ser Leu Gly Pro Glu Asp Glu Gly Val Tyr His Cys Ala Pro

100 105 110

Ser Ala Trp Val Gln His Ala Asp Tyr Ser Trp Tyr Gln Ala Gly Ser

115 120 125

Ala Arg Ser Gly Pro Val Thr Val Tyr Pro Tyr Met His Ala Leu Asp

130 135 140

Thr Leu Phe Val Pro Leu Leu Val Gly Thr Gly Val Ala Leu Val Thr

145 150 155 160

Gly Ala Thr Val Leu Gly Thr Ile Thr Cys Cys Phe Met Lys Arg Leu

165 170 175

Arg Lys Arg

<210> 200

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 200

Val Ala Leu Val Thr Gly Ala Thr Val Leu Gly Thr Ile Thr Cys Cys

1 5 10 15

Phe Met Lys Arg Leu Arg Lys Arg

<210> 201

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 201

Met Gly Ala Leu Arg Pro Thr Leu Leu Pro Pro Ser Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Met Leu Gly Met Gly Cys Trp Ala

20 25

<210> 202

<211> 1021

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 202

Met Ala Gly Ile Ser Tyr Val Ala Ser Phe Phe Leu Leu Leu Thr Lys

1 5 10 15

Leu Ser Ile Gly Gln Arg Glu Val Thr Val Gln Lys Gly Pro Leu Phe

20 25 30

Arg Ala Glu Gly Tyr Pro Val Ser Ile Gly Cys Asn Val Thr Gly His

35 40 45

Gln Gly Pro Ser Glu Gln His Phe Gln Trp Ser Val Tyr Leu Pro Thr

50 55 60

Asn Pro Thr Gln Glu Val Gln Ile Ile Ser Thr Lys Asp Ala Ala Phe

65 70 75 80

Ser Tyr Ala Val Tyr Thr Gln Arg Val Arg Ser Gly Asp Val Tyr Val

85 90 95

Glu Arg Val Gln Gly Asn Ser Val Leu Leu His Ile Ser Lys Leu Gln

100 105 110

Met Lys Asp Ala Gly Glu Tyr Glu Cys His Thr Pro Asn Thr Asp Glu

115 120 125

Lys Tyr Tyr Gly Ser Tyr Ser Ala Lys Thr Asn Leu Ile Val Ile Pro

130 135 140

Asp Thr Leu Ser Ala Thr Met Ser Ser Gln Thr Leu Gly Lys Glu Glu

145 150 155 160

Gly Glu Pro Leu Ala Leu Thr Cys Glu Ala Ser Lys Ala Thr Ala Gln

165 170 175

His Thr His Leu Ser Val Thr Trp Tyr Leu Thr Gln Asp Gly Gly Gly

180 185 190

Ser Gln Ala Thr Glu Ile Ile Ser Leu Ser Lys Asp Phe Ile Leu Val

195 200 205

Pro Gly Pro Leu Tyr Thr Glu Arg Phe Ala Ala Ser Asp Val Gln Leu

210 215 220

Asn Lys Leu Gly Pro Thr Thr Phe Arg Leu Ser Ile Glu Arg Leu Gln

225 230 235 240

Ser Ser Asp Gln Gly Gln Leu Phe Cys Glu Ala Thr Glu Trp Ile Gln

245 250 255

Asp Pro Asp Glu Thr Trp Met Phe Ile Thr Lys Lys Gln Thr Asp Gln

260 265 270

Thr Thr Leu Arg Ile Gln Pro Ala Val Lys Asp Phe Gln Val Asn Ile

275 280 285

Thr Ala Asp Ser Leu Phe Ala Glu Gly Lys Pro Leu Glu Leu Val Cys

290 295 300

Leu Val Val Ser Ser Gly Arg Asp Pro Gln Leu Gln Gly Ile Trp Phe

305 310 315 320

Phe Asn Gly Thr Glu Ile Ala His Ile Asp Ala Gly Gly Val Leu Gly

325 330 335

Leu Lys Asn Asp Tyr Lys Glu Arg Ala Ser Gln Gly Glu Leu Gln Val

340 345 350

Ser Lys Leu Gly Pro Lys Ala Phe Ser Leu Lys Ile Phe Ser Leu Gly

355 360 365

Pro Glu Asp Glu Gly Ala Tyr Arg Cys Val Val Ala Glu Val Met Lys

370 375 380

Thr Arg Thr Gly Ser Trp Gln Val Leu Gln Arg Lys Gln Ser Pro Asp

385 390 395 400

Ser His Val His Leu Arg Lys Pro Ala Ala Arg Ser Val Val Met Ser

405 410 415

Thr Lys Asn Lys Gln Gln Val Val Trp Glu Gly Glu Thr Leu Ala Phe

420 425 430

Leu Cys Lys Ala Gly Gly Ala Glu Ser Pro Leu Ser Val Ser Trp Trp

435 440 445

His Ile Pro Arg Asp Gln Thr Gln Pro Glu Phe Val Ala Gly Met Gly

450 455 460

Gln Asp Gly Ile Val Gln Leu Gly Ala Ser Tyr Gly Val Pro Ser Tyr

465 470 475 480

His Gly Asn Thr Arg Leu Glu Lys Met Asp Trp Ala Thr Phe Gln Leu

485 490 495

Glu Ile Thr Phe Thr Ala Ile Thr Asp Ser Gly Thr Tyr Glu Cys Arg

500 505 510

Val Ser Glu Lys Ser Arg Asn Gln Ala Arg Asp Leu Ser Trp Thr Gln

515 520 525

Lys Ile Ser Val Thr Val Lys Ser Leu Glu Ser Ser Leu Gln Val Ser

530 535 540

Leu Met Ser Arg Gln Pro Gln Val Met Leu Thr Asn Thr Phe Asp Leu

545 550 555 560

Ser Cys Val Val Arg Ala Gly Tyr Ser Asp Leu Lys Val Pro Leu Thr

565 570 575

Val Thr Trp Gln Phe Gln Pro Ala Ser Ser His Ile Phe His Gln Leu

580 585 590

Ile Arg Ile Thr His Asn Gly Thr Ile Glu Trp Gly Asn Phe Leu Ser

595 600 605

Arg Phe Gln Lys Lys Thr Lys Val Ser Gln Ser Leu Phe Arg Ser Gln

610 615 620

Leu Leu Val His Asp Ala Thr Glu Glu Glu Thr Gly Val Tyr Gln Cys

625 630 635 640

Glu Val Glu Val Tyr Asp Arg Asn Ser Leu Tyr Asn Asn Arg Pro Pro

645 650 655

Arg Ala Ser Ala Ile Ser His Pro Leu Arg Ile Ala Val Thr Leu Pro

660 665 670

Glu Ser Lys Leu Lys Val Asn Ser Arg Ser Gln Val Gln Glu Leu Ser

675 680 685

Ile Asn Ser Asn Thr Asp Ile Glu Cys Ser Ile Leu Ser Arg Ser Asn

690 695 700

Gly Asn Leu Gln Leu Ala Ile Ile Trp Tyr Phe Ser Pro Val Ser Thr

705 710 715 720

Asn Ala Ser Trp Leu Lys Ile Leu Glu Met Asp Gln Thr Asn Val Ile

725 730 735

Lys Thr Gly Asp Glu Phe His Thr Pro Gln Arg Lys Gln Lys Phe His

740 745 750

Thr Glu Lys Val Ser Gln Asp Leu Phe Gln Leu His Ile Leu Asn Val

755 760 765

Glu Asp Ser Asp Arg Gly Lys Tyr His Cys Ala Val Glu Glu Trp Leu

770 775 780

Leu Ser Thr Asn Gly Thr Trp His Lys Leu Gly Glu Lys Lys Ser Gly

785 790 795 800

Leu Thr Glu Leu Lys Leu Lys Pro Thr Gly Ser Lys Val Arg Val Ser

805 810 815

Lys Val Tyr Trp Thr Glu Asn Val Thr Glu His Arg Glu Val Ala Ile

820 825 830

Arg Cys Ser Leu Glu Ser Val Gly Ser Ser Ala Thr Leu Tyr Ser Val

835 840 845

Met Trp Tyr Trp Asn Arg Glu Asn Ser Gly Ser Lys Leu Leu Val His

850 855 860

Leu Gln His Asp Gly Leu Leu Glu Tyr Gly Glu Glu Gly Leu Arg Arg

865 870 875 880

His Leu His Cys Tyr Arg Ser Ser Ser Thr Asp Phe Val Leu Lys Leu

885 890 895

His Gln Val Glu Met Glu Asp Ala Gly Met Tyr Trp Cys Arg Val Ala

900 905 910

Glu Trp Gln Leu His Gly His Pro Ser Lys Trp Ile Asn Gln Ala Ser

915 920 925

Asp Glu Ser Gln Arg Met Val Leu Thr Val Leu Pro Ser Glu Pro Thr

930 935 940

Leu Pro Ser Arg Ile Cys Ser Ser Ala Pro Leu Leu Tyr Phe Leu Phe

945 950 955 960

Ile Cys Pro Phe Val Leu Leu Leu Leu Leu Leu Ile Ser Leu Leu Cys

965 970 975

Leu Tyr Trp Lys Ala Arg Lys Leu Ser Thr Leu Arg Ser Asn Thr Arg

980 985 990

Lys Glu Lys Ala Leu Trp Val Asp Leu Lys Glu Ala Gly Gly Val Thr

995 1000 1005

Thr Asn Arg Arg Glu Asp Glu Glu Glu Asp Glu Gly Asn

1010 1015 1020

<210> 203

<211> 1195

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 203

Met Lys Cys Phe Phe Pro Val Leu Ser Cys Leu Ala Val Leu Gly Val

1 5 10 15

Val Ser Ala Gln Arg Gln Val Thr Val Gln Glu Gly Pro Leu Tyr Arg

20 25 30

Thr Glu Gly Ser His Ile Thr Ile Trp Cys Asn Val Ser Gly Tyr Gln

35 40 45

Gly Pro Ser Glu Gln Asn Phe Gln Trp Ser Ile Tyr Leu Pro Ser Ser

50 55 60

Pro Glu Arg Glu Val Gln Ile Val Ser Thr Met Asp Ser Ser Phe Pro

65 70 75 80

Tyr Ala Ile Tyr Thr Gln Arg Val Arg Gly Gly Lys Ile Phe Ile Glu

85 90 95

Arg Val Gln Gly Asn Ser Thr Leu Leu His Ile Thr Asp Leu Gln Ala

100 105 110

Arg Asp Ala Gly Glu Tyr Glu Cys His Thr Pro Ser Thr Asp Lys Gln

115 120 125

Tyr Phe Gly Ser Tyr Ser Ala Lys Met Asn Leu Val Val Ile Pro Asp

130 135 140

Ser Leu Gln Thr Thr Ala Met Pro Gln Thr Leu His Arg Val Glu Gln

145 150 155 160

Asp Pro Leu Glu Leu Thr Cys Glu Val Ala Ser Glu Thr Ile Gln His

165 170 175

Ser His Leu Ser Val Ala Trp Leu Arg Gln Lys Val Gly Glu Lys Pro

180 185 190

Val Glu Val Ile Ser Leu Ser Arg Asp Phe Met Leu His Ser Ser Ser

195 200 205

Glu Tyr Ala Gln Arg Gln Ser Leu Gly Glu Val Arg Leu Asp Lys Leu

210 215 220

Gly Arg Thr Thr Phe Arg Leu Thr Ile Phe His Leu Gln Pro Ser Asp

225 230 235 240

Gln Gly Glu Phe Tyr Cys Glu Ala Ala Glu Trp Ile Gln Asp Pro Asp

245 250 255

Gly Ser Trp Tyr Ala Met Thr Arg Lys Arg Ser Glu Gly Ala Val Val

260 265 270

Asn Val Gln Pro Thr Asp Lys Glu Phe Thr Val Arg Leu Glu Thr Glu

275 280 285

Lys Arg Leu His Thr Val Gly Glu Pro Val Glu Phe Arg Cys Ile Leu

290 295 300

Glu Ala Gln Asn Val Pro Asp Arg Tyr Phe Ala Val Ser Trp Ala Phe

305 310 315 320

Asn Ser Ser Leu Ile Ala Thr Met Gly Pro Asn Ala Val Pro Val Leu

325 330 335

Asn Ser Glu Phe Ala His Arg Glu Ala Arg Gly Gln Leu Lys Val Ala

340 345 350

Lys Glu Ser Asp Ser Val Phe Val Leu Lys Ile Tyr His Leu Arg Gln

355 360 365

Glu Asp Ser Gly Lys Tyr Asn Cys Arg Val Thr Glu Arg Glu Lys Thr

370 375 380

Val Thr Gly Glu Phe Ile Asp Lys Glu Ser Lys Arg Pro Lys Asn Ile

385 390 395 400

Pro Ile Ile Val Leu Pro Leu Lys Ser Ser Ile Ser Val Glu Val Ala

405 410 415

Ser Asn Ala Ser Val Ile Leu Glu Gly Glu Asp Leu Arg Phe Ser Cys

420 425 430

Ser Val Arg Thr Ala Gly Arg Pro Gln Gly Arg Phe Ser Val Ile Trp

435 440 445

Gln Leu Val Asp Arg Gln Asn Arg Arg Ser Asn Ile Met Trp Leu Asp

450 455 460

Arg Asp Gly Thr Val Gln Pro Gly Ser Ser Tyr Trp Glu Arg Ser Ser

465 470 475 480

Phe Gly Gly Val Gln Met Glu Gln Val Gln Pro Asn Ser Phe Ser Leu

485 490 495

Gly Ile Phe Asn Ser Arg Lys Glu Asp Glu Gly Gln Tyr Glu Cys His

500 505 510

Val Thr Glu Trp Val Arg Ala Val Asp Gly Glu Trp Gln Ile Val Gly

515 520 525

Glu Arg Arg Ala Ser Thr Pro Ile Ser Ile Thr Ala Leu Glu Met Gly

530 535 540

Phe Ala Val Thr Ala Ile Ser Arg Thr Pro Gly Val Thr Tyr Ser Asp

545 550 555 560

Ser Phe Asp Leu Gln Cys Ile Ile Lys Pro His Tyr Pro Ala Trp Val

565 570 575

Pro Val Ser Val Thr Trp Arg Phe Gln Pro Val Gly Thr Val Glu Phe

580 585 590

His Asp Leu Val Thr Phe Thr Arg Asp Gly Gly Val Gln Trp Gly Asp

595 600 605

Arg Ser Ser Ser Phe Arg Thr Arg Thr Ala Ile Glu Lys Ala Glu Ser

610 615 620

Ser Asn Asn Val Arg Leu Ser Ile Ser Arg Ala Ser Asp Thr Glu Ala

625 630 635 640

Gly Lys Tyr Gln Cys Val Ala Glu Leu Trp Arg Lys Asn Tyr Asn Asn

645 650 655

Thr Trp Thr Arg Leu Ala Glu Arg Thr Ser Asn Leu Leu Glu Ile Arg

660 665 670

Val Leu Gln Pro Val Thr Lys Leu Gln Val Ser Lys Ser Lys Arg Thr

675 680 685

Leu Thr Leu Val Glu Asn Lys Pro Ile Gln Leu Asn Cys Ser Val Lys

690 695 700

Ser Gln Thr Ser Gln Asn Ser His Phe Ala Val Leu Trp Tyr Val His

705 710 715 720

Lys Pro Ser Asp Ala Asp Gly Lys Leu Ile Leu Lys Thr Thr His Asn

725 730 735

Ser Ala Phe Glu Tyr Gly Thr Tyr Ala Glu Glu Glu Gly Leu Arg Ala

740 745 750

Arg Leu Gln Phe Glu Arg His Val Ser Gly Gly Leu Phe Ser Leu Thr

755 760 765

Val Gln Arg Ala Glu Val Ser Asp Ser Gly Ser Tyr Tyr Cys His Val

770 775 780

Glu Glu Trp Leu Leu Ser Pro Asn Tyr Ala Trp Tyr Lys Leu Ala Glu

785 790 795 800

Glu Val Ser Gly Arg Thr Glu Val Thr Val Lys Gln Pro Asp Ser Arg

805 810 815

Leu Arg Leu Ser Gln Ala Gln Gly Asn Leu Ser Val Leu Glu Thr Arg

820 825 830

Gln Val Gln Leu Glu Cys Val Val Leu Asn Arg Thr Ser Ile Thr Ser

835 840 845

Gln Leu Met Val Glu Trp Phe Val Trp Lys Pro Asn His Pro Glu Arg

850 855 860

Glu Thr Val Ala Arg Leu Ser Arg Asp Ala Thr Phe His Tyr Gly Glu

865 870 875 880

Gln Ala Ala Lys Asn Asn Leu Lys Gly Arg Leu His Leu Glu Ser Pro

885 890 895

Ser Pro Gly Val Tyr Arg Leu Phe Ile Gln Asn Val Ala Val Gln Asp

900 905 910

Ser Gly Thr Tyr Ser Cys His Val Glu Glu Trp Leu Pro Ser Pro Ser

915 920 925

Gly Met Trp Tyr Lys Arg Ala Glu Asp Thr Ala Gly Gln Thr Ala Leu

930 935 940

Thr Val Met Arg Pro Asp Ala Ser Leu Gln Val Asp Thr Val Val Pro

945 950 955 960

Asn Ala Thr Val Ser Glu Lys Ala Ala Phe Gln Leu Asp Cys Ser Ile

965 970 975

Val Ser Arg Ser Ser Gln Asp Ser Arg Phe Ala Val Ala Trp Tyr Ser

980 985 990

Leu Arg Thr Lys Ala Gly Gly Lys Arg Ser Ser Pro Gly Leu Glu Glu

995 1000 1005

Gln Glu Glu Glu Arg Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp

1010 1015 1020

Asp Asp Asp Asp Pro Thr Glu Arg Thr Ala Leu Leu Ser Val Gly

1025 1030 1035

Pro Asp Ala Val Phe Gly Pro Glu Gly Ser Pro Trp Glu Gly Arg

1040 1045 1050

Leu Arg Phe Gln Arg Leu Ser Pro Val Leu Tyr Arg Leu Thr Val

1055 1060 1065

Leu Gln Ala Ser Pro Gln Asp Thr Gly Asn Tyr Ser Cys His Val

1070 1075 1080

Glu Glu Trp Leu Pro Ser Pro Gln Lys Glu Trp Tyr Arg Leu Thr

1085 1090 1095

Glu Glu Glu Ser Ala Pro Ile Gly Ile Arg Val Leu Asp Thr Ser

1100 1105 1110

Pro Thr Leu Gln Ser Ile Ile Cys Ser Asn Asp Ala Leu Phe Tyr

1115 1120 1125

Phe Val Phe Phe Tyr Pro Phe Pro Ile Phe Gly Ile Leu Ile Ile

1130 1135 1140

Thr Ile Leu Leu Val Arg Phe Lys Ser Arg Asn Ser Ser Lys Asn

1145 1150 1155

Ser Asp Gly Lys Asn Gly Val Pro Leu Leu Trp Ile Lys Glu Pro

1160 1165 1170

His Leu Asn Tyr Ser Pro Thr Cys Leu Glu Pro Pro Val Leu Ser

1175 1180 1185

Ile His Pro Gly Ala Ile Asp

1190 1195

<210> 204

<211> 1023

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 204

Met Gly Lys Gly Val Gly Arg Asp Lys Tyr Glu Pro Ala Ala Val Ser

1 5 10 15

Glu Gln Gly Asp Lys Lys Gly Lys Lys Gly Lys Lys Asp Arg Asp Met

20 25 30

Asp Glu Leu Lys Lys Glu Val Ser Met Asp Asp His Lys Leu Ser Leu

35 40 45

Asp Glu Leu His Arg Lys Tyr Gly Thr Asp Leu Ser Arg Gly Leu Thr

50 55 60

Ser Ala Arg Ala Ala Glu Ile Leu Ala Arg Asp Gly Pro Asn Ala Leu

65 70 75 80

Thr Pro Pro Pro Thr Thr Pro Glu Trp Ile Lys Phe Cys Arg Gln Leu

85 90 95

Phe Gly Gly Phe Ser Met Leu Leu Trp Ile Gly Ala Ile Leu Cys Phe

100 105 110

Leu Ala Tyr Ser Ile Gln Ala Ala Thr Glu Glu Glu Pro Gln Asn Asp

115 120 125

Asn Leu Tyr Leu Gly Val Val Leu Ser Ala Val Val Ile Ile Thr Gly

130 135 140

Cys Phe Ser Tyr Tyr Gln Glu Ala Lys Ser Ser Lys Ile Met Glu Ser

145 150 155 160

Phe Lys Asn Met Val Pro Gln Gln Ala Leu Val Ile Arg Asn Gly Glu

165 170 175

Lys Met Ser Ile Asn Ala Glu Glu Val Val Val Gly Asp Leu Val Glu

180 185 190

Val Lys Gly Gly Asp Arg Ile Pro Ala Asp Leu Arg Ile Ile Ser Ala

195 200 205

Asn Gly Cys Lys Val Asp Asn Ser Ser Leu Thr Gly Glu Ser Glu Pro

210 215 220

Gln Thr Arg Ser Pro Asp Phe Thr Asn Glu Asn Pro Leu Glu Thr Arg

225 230 235 240

Asn Ile Ala Phe Phe Ser Thr Asn Cys Val Glu Gly Thr Ala Arg Gly

245 250 255

Ile Val Val Tyr Thr Gly Asp Arg Thr Val Met Gly Arg Ile Ala Thr

260 265 270

Leu Ala Ser Gly Leu Glu Gly Gly Gln Thr Pro Ile Ala Ala Glu Ile

275 280 285

Glu His Phe Ile His Ile Ile Thr Gly Val Ala Val Phe Leu Gly Val

290 295 300

Ser Phe Phe Ile Leu Ser Leu Ile Leu Glu Tyr Thr Trp Leu Glu Ala

305 310 315 320

Val Ile Phe Leu Ile Gly Ile Ile Val Ala Asn Val Pro Glu Gly Leu

325 330 335

Leu Ala Thr Val Thr Val Cys Leu Thr Leu Thr Ala Lys Arg Met Ala

340 345 350

Arg Lys Asn Cys Leu Val Lys Asn Leu Glu Ala Val Glu Thr Leu Gly

355 360 365

Ser Thr Ser Thr Ile Cys Ser Asp Lys Thr Gly Thr Leu Thr Gln Asn

370 375 380

Arg Met Thr Val Ala His Met Trp Phe Asp Asn Gln Ile His Glu Ala

385 390 395 400

Asp Thr Thr Glu Asn Gln Ser Gly Val Ser Phe Asp Lys Thr Ser Ala

405 410 415

Thr Trp Leu Ala Leu Ser Arg Ile Ala Gly Leu Cys Asn Arg Ala Val

420 425 430

Phe Gln Ala Asn Gln Glu Asn Leu Pro Ile Leu Lys Arg Ala Val Ala

435 440 445

Gly Asp Ala Ser Glu Ser Ala Leu Leu Lys Cys Ile Glu Leu Cys Cys

450 455 460

Gly Ser Val Lys Glu Met Arg Glu Arg Tyr Ala Lys Ile Val Glu Ile

465 470 475 480

Pro Phe Asn Ser Thr Asn Lys Tyr Gln Leu Ser Ile His Lys Asn Pro

485 490 495

Asn Thr Ser Glu Pro Gln His Leu Leu Val Met Lys Gly Ala Pro Glu

500 505 510

Arg Ile Leu Asp Arg Cys Ser Ser Ile Leu Leu His Gly Lys Glu Gln

515 520 525

Pro Leu Asp Glu Glu Leu Lys Asp Ala Phe Gln Asn Ala Tyr Leu Glu

530 535 540

Leu Gly Gly Leu Gly Glu Arg Val Leu Gly Phe Cys His Leu Phe Leu

545 550 555 560

Pro Asp Glu Gln Phe Pro Glu Gly Phe Gln Phe Asp Thr Asp Asp Val

565 570 575

Asn Phe Pro Ile Asp Asn Leu Cys Phe Val Gly Leu Ile Ser Met Ile

580 585 590

Asp Pro Pro Arg Ala Ala Val Pro Asp Ala Val Gly Lys Cys Arg Ser

595 600 605

Ala Gly Ile Lys Val Ile Met Val Thr Gly Asp His Pro Ile Thr Ala

610 615 620

Lys Ala Ile Ala Lys Gly Val Gly Ile Ile Ser Glu Gly Asn Glu Thr

625 630 635 640

Val Glu Asp Ile Ala Ala Arg Leu Asn Ile Pro Val Ser Gln Val Asn

645 650 655

Pro Arg Asp Ala Lys Ala Cys Val Val His Gly Ser Asp Leu Lys Asp

660 665 670

Met Thr Ser Glu Gln Leu Asp Asp Ile Leu Lys Tyr His Thr Glu Ile

675 680 685

Val Phe Ala Arg Thr Ser Pro Gln Gln Lys Leu Ile Ile Val Glu Gly

690 695 700

Cys Gln Arg Gln Gly Ala Ile Val Ala Val Thr Gly Asp Gly Val Asn

705 710 715 720

Asp Ser Pro Ala Leu Lys Lys Ala Asp Ile Gly Val Ala Met Gly Ile

725 730 735

Ala Gly Ser Asp Val Ser Lys Gln Ala Ala Asp Met Ile Leu Leu Asp

740 745 750

Asp Asn Phe Ala Ser Ile Val Thr Gly Val Glu Glu Gly Arg Leu Ile

755 760 765

Phe Asp Asn Leu Lys Lys Ser Ile Ala Tyr Thr Leu Thr Ser Asn Ile

770 775 780

Pro Glu Ile Thr Pro Phe Leu Ile Phe Ile Ile Ala Asn Ile Pro Leu

785 790 795 800

Pro Leu Gly Thr Val Thr Ile Leu Cys Ile Asp Leu Gly Thr Asp Met

805 810 815

Val Pro Ala Ile Ser Leu Ala Tyr Glu Gln Ala Glu Ser Asp Ile Met

820 825 830

Lys Arg Gln Pro Arg Asn Pro Lys Thr Asp Lys Leu Val Asn Glu Arg

835 840 845

Leu Ile Ser Met Ala Tyr Gly Gln Ile Gly Met Ile Gln Ala Leu Gly

850 855 860

Gly Phe Phe Thr Tyr Phe Val Ile Leu Ala Glu Asn Gly Phe Leu Pro

865 870 875 880

Ile His Leu Leu Gly Leu Arg Val Asp Trp Asp Asp Arg Trp Ile Asn

885 890 895

Asp Val Glu Asp Ser Tyr Gly Gln Gln Trp Thr Tyr Glu Gln Arg Lys

900 905 910

Ile Val Glu Phe Thr Cys His Thr Ala Phe Phe Val Ser Ile Val Val

915 920 925

Val Gln Trp Ala Asp Leu Val Ile Cys Lys Thr Arg Arg Asn Ser Val

930 935 940

Phe Gln Gln Gly Met Lys Asn Lys Ile Leu Ile Phe Gly Leu Phe Glu

945 950 955 960

Glu Thr Ala Leu Ala Ala Phe Leu Ser Tyr Cys Pro Gly Met Gly Val

965 970 975

Ala Leu Arg Met Tyr Pro Leu Lys Pro Thr Trp Trp Phe Cys Ala Phe

980 985 990

Pro Tyr Ser Leu Leu Ile Phe Val Tyr Asp Glu Val Arg Lys Leu Ile

995 1000 1005

Ile Arg Arg Arg Pro Gly Gly Trp Val Glu Lys Glu Thr Tyr Tyr

1010 1015 1020

<210> 205

<211> 240

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 205

Met Gly Arg Gly Ala Gly Arg Glu Tyr Ser Pro Ala Ala Thr Thr Ala

1 5 10 15

Glu Asn Gly Gly Gly Lys Lys Lys Gln Lys Glu Lys Glu Leu Asp Glu

20 25 30

Leu Lys Lys Glu Val Ala Met Asp Asp His Lys Leu Ser Leu Asp Glu

35 40 45

Leu Gly Arg Lys Tyr Gln Val Asp Leu Ser Lys Gly Leu Thr Asn Gln

50 55 60

Arg Ala Gln Asp Val Leu Ala Arg Asp Gly Pro Asn Ala Leu Thr Pro

65 70 75 80

Pro Pro Thr Thr Pro Glu Trp Val Lys Phe Cys Arg Gln Leu Phe Gly

85 90 95

Gly Phe Ser Ile Leu Leu Trp Ile Gly Ala Ile Leu Cys Phe Leu Ala

100 105 110

Tyr Gly Ile Gln Ala Ala Met Glu Asp Glu Pro Ser Asn Asp Asn Leu

115 120 125

Tyr Leu Gly Val Val Leu Ala Ala Val Val Ile Val Thr Gly Cys Phe

130 135 140

Ser Tyr Tyr Gln Glu Ala Lys Ser Ser Lys Ile Met Asp Ser Phe Lys

145 150 155 160

Asn Met Val Pro Gln Gln Ala Leu Val Ile Arg Glu Gly Glu Lys Met

165 170 175

Gln Ile Asn Ala Glu Glu Val Val Val Gly Asp Leu Val Glu Val Lys

180 185 190

Gly Gly Asp Arg Val Pro Ala Asp Leu Arg Ile Ile Ser Ser His Gly

195 200 205

Cys Lys Val Asp Asn Ser Ser Leu Thr Gly Glu Ser Glu Pro Gln Thr

210 215 220

Arg Ser Pro Glu Phe Thr His Glu Asn Pro Leu Glu Thr Arg Asn Ile

225 230 235 240

<210> 206

<211> 780

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 206

Cys Phe Phe Ser Thr Asn Cys Val Glu Gly Thr Ala Arg Gly Ile Val

1 5 10 15

Ile Ala Thr Gly Asp Arg Thr Val Met Gly Arg Ile Ala Thr Leu Ala

20 25 30

Ser Gly Leu Glu Val Gly Arg Thr Pro Ile Ala Met Glu Ile Glu His

35 40 45

Phe Ile Gln Leu Ile Thr Gly Val Ala Val Phe Leu Gly Val Ser Phe

50 55 60

Phe Val Leu Ser Leu Ile Leu Gly Tyr Ser Trp Leu Glu Ala Val Ile

65 70 75 80

Phe Leu Ile Gly Ile Ile Val Ala Asn Val Pro Glu Gly Leu Leu Ala

85 90 95

Thr Val Thr Val Cys Leu Thr Leu Thr Ala Lys Arg Met Ala Arg Lys

100 105 110

Asn Cys Leu Val Lys Asn Leu Glu Ala Val Glu Thr Leu Gly Ser Thr

115 120 125

Ser Thr Ile Cys Ser Asp Lys Thr Gly Thr Leu Thr Gln Asn Arg Met

130 135 140

Thr Val Ala His Met Trp Phe Asp Asn Gln Ile His Glu Ala Asp Thr

145 150 155 160

Thr Glu Asp Gln Ser Gly Ala Thr Phe Asp Lys Arg Ser Pro Thr Trp

165 170 175

Thr Ala Leu Ser Arg Ile Ala Gly Leu Cys Asn Arg Ala Val Phe Lys

180 185 190

Ala Gly Gln Glu Asn Ile Ser Val Ser Lys Arg Asp Thr Ala Gly Asp

195 200 205

Ala Ser Glu Ser Ala Leu Leu Lys Cys Ile Glu Leu Ser Cys Gly Ser

210 215 220

Val Arg Lys Met Arg Asp Arg Asn Pro Lys Val Ala Glu Ile Pro Phe

225 230 235 240

Asn Ser Thr Asn Lys Tyr Gln Leu Ser Ile His Glu Arg Glu Asp Ser

245 250 255

Pro Gln Ser His Val Leu Val Met Lys Gly Ala Pro Glu Arg Ile Leu

260 265 270

Asp Arg Cys Ser Thr Ile Leu Val Gln Gly Lys Glu Ile Pro Leu Asp

275 280 285

Lys Glu Met Gln Asp Ala Phe Gln Asn Ala Tyr Met Glu Leu Gly Gly

290 295 300

Leu Gly Glu Arg Val Leu Gly Phe Cys Gln Leu Asn Leu Pro Ser Gly

305 310 315 320

Lys Phe Pro Arg Gly Phe Lys Phe Asp Thr Asp Glu Leu Asn Phe Pro

325 330 335

Thr Glu Lys Leu Cys Phe Val Gly Leu Met Ser Met Ile Asp Pro Pro

340 345 350

Arg Ala Ala Val Pro Asp Ala Val Gly Lys Cys Arg Ser Ala Gly Ile

355 360 365

Lys Val Ile Met Val Thr Gly Asp His Pro Ile Thr Ala Lys Ala Ile

370 375 380

Ala Lys Gly Val Gly Ile Ile Ser Glu Gly Asn Glu Thr Val Glu Asp

385 390 395 400

Ile Ala Ala Arg Leu Asn Ile Pro Met Ser Gln Val Asn Pro Arg Glu

405 410 415

Ala Lys Ala Cys Val Val His Gly Ser Asp Leu Lys Asp Met Thr Ser

420 425 430

Glu Gln Leu Asp Glu Ile Leu Lys Asn His Thr Glu Ile Val Phe Ala

435 440 445

Arg Thr Ser Pro Gln Gln Lys Leu Ile Ile Val Glu Gly Cys Gln Arg

450 455 460

Gln Gly Ala Ile Val Ala Val Thr Gly Asp Gly Val Asn Asp Ser Pro

465 470 475 480

Ala Leu Lys Lys Ala Asp Ile Gly Ile Ala Met Gly Ile Ser Gly Ser

485 490 495

Asp Val Ser Lys Gln Ala Ala Asp Met Ile Leu Leu Asp Asp Asn Phe

500 505 510

Ala Ser Ile Val Thr Gly Val Glu Glu Gly Arg Leu Ile Phe Asp Asn

515 520 525

Leu Lys Lys Ser Ile Ala Tyr Thr Leu Thr Ser Asn Ile Pro Glu Ile

530 535 540

Thr Pro Phe Leu Leu Phe Ile Ile Ala Asn Ile Pro Leu Pro Leu Gly

545 550 555 560

Thr Val Thr Ile Leu Cys Ile Asp Leu Gly Thr Asp Met Val Pro Ala

565 570 575

Ile Ser Leu Ala Tyr Glu Ala Ala Glu Ser Asp Ile Met Lys Arg Gln

580 585 590

Pro Arg Asn Ser Gln Thr Asp Lys Leu Val Asn Glu Arg Leu Ile Ser

595 600 605

Met Ala Tyr Gly Gln Ile Gly Met Ile Gln Ala Leu Gly Gly Phe Phe

610 615 620

Thr Tyr Phe Val Ile Leu Ala Glu Asn Gly Phe Leu Pro Ser Arg Leu

625 630 635 640

Leu Gly Ile Arg Leu Asp Trp Asp Asp Arg Thr Met Asn Asp Leu Glu

645 650 655

Asp Ser Tyr Gly Gln Glu Trp Thr Tyr Glu Gln Arg Lys Val Val Glu

660 665 670

Phe Thr Cys His Thr Ala Phe Phe Ala Ser Ile Val Val Val Gln Trp

675 680 685

Ala Asp Leu Ile Ile Cys Lys Thr Arg Arg Asn Ser Val Phe Gln Gln

690 695 700

Gly Met Lys Asn Lys Ile Leu Ile Phe Gly Leu Leu Glu Glu Thr Ala

705 710 715 720

Leu Ala Ala Phe Leu Ser Tyr Cys Pro Gly Met Gly Val Ala Leu Arg

725 730 735

Met Tyr Pro Leu Lys Val Thr Trp Trp Phe Cys Ala Phe Pro Tyr Ser

740 745 750

Leu Leu Ile Phe Ile Tyr Asp Glu Val Arg Lys Leu Ile Leu Arg Arg

755 760 765

Tyr Pro Gly Gly Trp Val Glu Lys Glu Thr Tyr Tyr

770 775 780

<210> 207

<211> 1026

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 207

Met Gly Ser Gly Gly Ser Asp Ser Tyr Arg Ile Ala Thr Ser Gln Asp

1 5 10 15

Lys Lys Asp Asp Lys Asp Ser Pro Lys Lys Asn Lys Gly Lys Glu Arg

20 25 30

Arg Asp Leu Asp Asp Leu Lys Lys Glu Val Ala Met Thr Glu His Lys

35 40 45

Met Ser Val Glu Glu Val Cys Arg Lys Tyr Asn Thr Asp Cys Val Gln

50 55 60

Gly Leu Thr His Ser Lys Ala Gln Glu Ile Leu Ala Arg Asp Gly Pro

65 70 75 80

Asn Ala Leu Thr Pro Pro Pro Thr Thr Pro Glu Trp Val Lys Phe Cys

85 90 95

Arg Gln Leu Phe Gly Gly Phe Ser Ile Leu Leu Trp Ile Gly Ala Ile

100 105 110

Leu Cys Phe Leu Ala Tyr Gly Ile Gln Ala Gly Thr Glu Asp Asp Pro

115 120 125

Ser Gly Asp Asn Leu Tyr Leu Gly Ile Val Leu Ala Ala Val Val Ile

130 135 140

Ile Thr Gly Cys Phe Ser Tyr Tyr Gln Glu Ala Lys Ser Ser Lys Ile

145 150 155 160

Met Glu Ser Phe Lys Asn Met Val Pro Gln Gln Ala Leu Val Ile Arg

165 170 175

Glu Gly Glu Lys Met Gln Val Asn Ala Glu Glu Val Val Val Gly Asp

180 185 190

Leu Val Glu Ile Lys Gly Gly Asp Arg Val Pro Ala Asp Leu Arg Ile

195 200 205

Ile Ser Ala His Gly Cys Lys Val Asp Asn Ser Ser Leu Thr Gly Glu

210 215 220

Ser Glu Pro Gln Thr Arg Ser Pro Asp Cys Thr His Asp Asn Pro Leu

225 230 235 240

Glu Thr Arg Asn Ile Thr Phe Phe Ser Thr Asn Cys Val Glu Gly Thr

245 250 255

Ala Arg Gly Val Val Val Ala Thr Gly Asp Arg Thr Val Met Gly Arg

260 265 270

Ile Ala Thr Leu Ala Ser Gly Leu Glu Val Gly Lys Thr Pro Ile Ala

275 280 285

Ile Glu Ile Glu His Phe Ile Gln Leu Ile Thr Gly Val Ala Val Phe

290 295 300

Leu Gly Val Ser Phe Phe Ile Leu Ser Leu Ile Leu Gly Tyr Thr Trp

305 310 315 320

Leu Glu Ala Val Ile Phe Leu Ile Gly Ile Ile Val Ala Asn Val Pro

325 330 335

Glu Gly Leu Leu Ala Thr Val Thr Val Cys Leu Thr Leu Thr Ala Lys

340 345 350

Arg Met Ala Arg Lys Asn Cys Leu Val Lys Asn Leu Glu Ala Val Glu

355 360 365

Thr Leu Gly Ser Thr Ser Thr Ile Cys Ser Asp Lys Thr Gly Thr Leu

370 375 380

Thr Gln Asn Arg Met Thr Val Ala His Met Trp Phe Asp Asn Gln Ile

385 390 395 400

His Glu Ala Asp Thr Thr Glu Asp Gln Ser Gly Thr Ser Phe Asp Lys

405 410 415

Ser Ser His Thr Trp Val Ala Leu Ser His Ile Ala Gly Leu Cys Asn

420 425 430

Arg Ala Val Phe Lys Gly Gly Gln Asp Asn Ile Pro Val Leu Lys Arg

435 440 445

Asp Val Ala Gly Asp Ala Ser Glu Ser Ala Leu Leu Lys Cys Ile Glu

450 455 460

Leu Ser Ser Gly Ser Val Lys Leu Met Arg Glu Arg Asn Lys Lys Val

465 470 475 480

Ala Glu Ile Pro Phe Asn Ser Thr Asn Lys Tyr Gln Leu Ser Ile His

485 490 495

Glu Thr Glu Asp Pro Asn Asp Asn Arg Tyr Leu Leu Val Met Lys Gly

500 505 510

Ala Pro Glu Arg Ile Leu Asp Arg Cys Ser Thr Ile Leu Leu Gln Gly

515 520 525

Lys Glu Gln Pro Leu Asp Glu Glu Met Lys Glu Ala Phe Gln Asn Ala

530 535 540

Tyr Leu Glu Leu Gly Gly Leu Gly Glu Arg Val Leu Gly Phe Cys His

545 550 555 560

Tyr Tyr Leu Pro Glu Glu Gln Phe Pro Lys Gly Phe Ala Phe Asp Cys

565 570 575

Asp Asp Val Asn Phe Thr Thr Asp Asn Leu Cys Phe Val Gly Leu Met

580 585 590

Ser Met Ile Asp Pro Pro Arg Ala Ala Val Pro Asp Ala Val Gly Lys

595 600 605

Cys Arg Ser Ala Gly Ile Lys Val Ile Met Val Thr Gly Asp His Pro

610 615 620

Ile Thr Ala Lys Ala Ile Ala Lys Gly Val Gly Ile Ile Ser Glu Gly

625 630 635 640

Asn Glu Thr Val Glu Asp Ile Ala Ala Arg Leu Asn Ile Pro Val Ser

645 650 655

Gln Val Asn Pro Arg Asp Ala Lys Ala Cys Val Ile His Gly Thr Asp

660 665 670

Leu Lys Asp Phe Thr Ser Glu Gln Ile Asp Glu Ile Leu Gln Asn His

675 680 685

Thr Glu Ile Val Phe Ala Arg Thr Ser Pro Gln Gln Lys Leu Ile Ile

690 695 700

Val Glu Gly Cys Gln Arg Gln Gly Ala Ile Val Ala Val Thr Gly Asp

705 710 715 720

Gly Val Asn Asp Ser Pro Ala Leu Lys Lys Ala Asp Ile Gly Val Ala

725 730 735

Met Gly Ile Ala Gly Ser Asp Val Ser Lys Gln Ala Ala Asp Met Ile

740 745 750

Leu Leu Asp Asp Asn Phe Ala Ser Ile Val Thr Gly Val Glu Glu Gly

755 760 765

Arg Leu Ile Phe Asp Asn Leu Lys Lys Ser Ile Ala Tyr Thr Leu Thr

770 775 780

Ser Asn Ile Pro Glu Ile Thr Pro Phe Leu Leu Phe Ile Met Ala Asn

785 790 795 800

Ile Pro Leu Pro Leu Gly Thr Ile Thr Ile Leu Cys Ile Asp Leu Gly

805 810 815

Thr Asp Met Val Pro Ala Ile Ser Leu Ala Tyr Glu Ala Ala Glu Ser

820 825 830

Asp Ile Met Lys Arg Gln Pro Arg Asn Pro Arg Thr Asp Lys Leu Val

835 840 845

Asn Glu Arg Leu Ile Ser Met Ala Tyr Gly Gln Ile Gly Met Ile Gln

850 855 860

Ala Leu Gly Gly Phe Phe Ser Tyr Phe Val Ile Leu Ala Glu Asn Gly

865 870 875 880

Phe Leu Pro Gly Asn Leu Val Gly Ile Arg Leu Asn Trp Asp Asp Arg

885 890 895

Thr Val Asn Asp Leu Glu Asp Ser Tyr Gly Gln Gln Trp Thr Tyr Glu

900 905 910

Gln Arg Lys Val Val Glu Phe Thr Cys His Thr Ala Phe Phe Val Ser

915 920 925

Ile Val Val Val Gln Trp Ala Asp Leu Ile Ile Cys Lys Thr Arg Arg

930 935 940

Asn Ser Val Phe Gln Gln Gly Met Lys Asn Lys Ile Leu Ile Phe Gly

945 950 955 960

Leu Phe Glu Glu Thr Ala Leu Ala Ala Phe Leu Ser Tyr Cys Pro Gly

965 970 975

Met Asp Val Ala Leu Arg Met Tyr Pro Leu Lys Pro Ser Trp Trp Phe

980 985 990

Cys Ala Phe Pro Tyr Ser Phe Leu Ile Phe Val Tyr Asp Glu Ile Arg

995 1000 1005

Lys Leu Ile Leu Arg Arg Asn Pro Gly Gly Trp Val Glu Lys Glu

1010 1015 1020

Thr Tyr Tyr

1025

<210> 208

<211> 1029

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 208

Met Gly Leu Trp Gly Lys Lys Gly Thr Val Ala Pro His Asp Gln Ser

1 5 10 15

Pro Arg Arg Arg Pro Lys Lys Gly Leu Ile Lys Lys Lys Met Val Lys

20 25 30

Arg Glu Lys Gln Lys Arg Asn Met Glu Glu Leu Lys Lys Glu Val Val

35 40 45

Met Asp Asp His Lys Leu Thr Leu Glu Glu Leu Ser Thr Lys Tyr Ser

50 55 60

Val Asp Leu Thr Lys Gly His Ser His Gln Arg Ala Lys Glu Ile Leu

65 70 75 80

Thr Arg Gly Gly Pro Asn Thr Val Thr Pro Pro Pro Thr Thr Pro Glu

85 90 95

Trp Val Lys Phe Cys Lys Gln Leu Phe Gly Gly Phe Ser Leu Leu Leu

100 105 110

Trp Thr Gly Ala Ile Leu Cys Phe Val Ala Tyr Ser Ile Gln Ile Tyr

115 120 125

Phe Asn Glu Glu Pro Thr Lys Asp Asn Leu Tyr Leu Ser Ile Val Leu

130 135 140

Ser Val Val Val Ile Val Thr Gly Cys Phe Ser Tyr Tyr Gln Glu Ala

145 150 155 160

Lys Ser Ser Lys Ile Met Glu Ser Phe Lys Asn Met Val Pro Gln Gln

165 170 175

Ala Leu Val Ile Arg Gly Gly Glu Lys Met Gln Ile Asn Val Gln Glu

180 185 190

Val Val Leu Gly Asp Leu Val Glu Ile Lys Gly Gly Asp Arg Val Pro

195 200 205

Ala Asp Leu Arg Leu Ile Ser Ala Gln Gly Cys Lys Val Asp Asn Ser

210 215 220

Ser Leu Thr Gly Glu Ser Glu Pro Gln Ser Arg Ser Pro Asp Phe Thr

225 230 235 240

His Glu Asn Pro Leu Glu Thr Arg Asn Ile Cys Phe Phe Ser Thr Asn

245 250 255

Cys Val Glu Gly Thr Ala Arg Gly Ile Val Ile Ala Thr Gly Asp Ser

260 265 270

Thr Val Met Gly Arg Ile Ala Ser Leu Thr Ser Gly Leu Ala Val Gly

275 280 285

Gln Thr Pro Ile Ala Ala Glu Ile Glu His Phe Ile His Leu Ile Thr

290 295 300

Val Val Ala Val Phe Leu Gly Val Thr Phe Phe Ala Leu Ser Leu Leu

305 310 315 320

Leu Gly Tyr Gly Trp Leu Glu Ala Ile Ile Phe Leu Ile Gly Ile Ile

325 330 335

Val Ala Asn Val Pro Glu Gly Leu Leu Ala Thr Val Thr Val Cys Leu

340 345 350

Thr Leu Thr Ala Lys Arg Met Ala Arg Lys Asn Cys Leu Val Lys Asn

355 360 365

Leu Glu Ala Val Glu Thr Leu Gly Ser Thr Ser Thr Ile Cys Ser Asp

370 375 380

Lys Thr Gly Thr Leu Thr Gln Asn Arg Met Thr Val Ala His Met Trp

385 390 395 400

Phe Asp Met Thr Val Tyr Glu Ala Asp Thr Thr Glu Glu Gln Thr Gly

405 410 415

Lys Thr Phe Thr Lys Ser Ser Asp Thr Trp Phe Met Leu Ala Arg Ile

420 425 430

Ala Gly Leu Cys Asn Arg Ala Asp Phe Lys Ala Asn Gln Glu Ile Leu

435 440 445

Pro Ile Ala Lys Arg Ala Thr Thr Gly Asp Ala Ser Glu Ser Ala Leu

450 455 460

Leu Lys Phe Ile Glu Gln Ser Tyr Ser Ser Val Ala Glu Met Arg Glu

465 470 475 480

Lys Asn Pro Lys Val Ala Glu Ile Pro Phe Asn Ser Thr Asn Lys Tyr

485 490 495

Gln Met Ser Ile His Leu Arg Glu Asp Ser Ser Gln Thr His Val Leu

500 505 510

Met Met Lys Gly Ala Pro Glu Arg Ile Leu Glu Phe Cys Ser Thr Phe

515 520 525

Leu Leu Asn Gly Gln Glu Tyr Ser Met Asn Asp Glu Met Lys Glu Ala

530 535 540

Phe Gln Asn Ala Tyr Leu Glu Leu Gly Gly Leu Gly Glu Arg Val Leu

545 550 555 560

Gly Phe Cys Phe Leu Asn Leu Pro Ser Ser Phe Ser Lys Gly Phe Pro

565 570 575

Phe Asn Thr Asp Glu Ile Asn Phe Pro Met Asp Asn Leu Cys Phe Val

580 585 590

Gly Leu Ile Ser Met Ile Asp Pro Pro Arg Ala Ala Val Pro Asp Ala

595 600 605

Val Ser Lys Cys Arg Ser Ala Gly Ile Lys Val Ile Met Val Thr Gly

610 615 620

Asp His Pro Ile Thr Ala Lys Ala Ile Ala Lys Gly Val Gly Ile Ile

625 630 635 640

Ser Glu Gly Thr Glu Thr Ala Glu Glu Val Ala Ala Arg Leu Lys Ile

645 650 655

Pro Ile Ser Lys Val Asp Ala Ser Ala Ala Lys Ala Ile Val Val His

660 665 670

Gly Ala Glu Leu Lys Asp Ile Gln Ser Lys Gln Leu Asp Gln Ile Leu

675 680 685

Gln Asn His Pro Glu Ile Val Phe Ala Arg Thr Ser Pro Gln Gln Lys

690 695 700

Leu Ile Ile Val Glu Gly Cys Gln Arg Leu Gly Ala Val Val Ala Val

705 710 715 720

Thr Gly Asp Gly Val Asn Asp Ser Pro Ala Leu Lys Lys Ala Asp Ile

725 730 735

Gly Ile Ala Met Gly Ile Ser Gly Ser Asp Val Ser Lys Gln Ala Ala

740 745 750

Asp Met Ile Leu Leu Asp Asp Asn Phe Ala Ser Ile Val Thr Gly Val

755 760 765

Glu Glu Gly Arg Leu Ile Phe Asp Asn Leu Lys Lys Ser Ile Met Tyr

770 775 780

Thr Leu Thr Ser Asn Ile Pro Glu Ile Thr Pro Phe Leu Met Phe Ile

785 790 795 800

Ile Leu Gly Ile Pro Leu Pro Leu Gly Thr Ile Thr Ile Leu Cys Ile

805 810 815

Asp Leu Gly Thr Asp Met Val Pro Ala Ile Ser Leu Ala Tyr Glu Ser

820 825 830

Ala Glu Ser Asp Ile Met Lys Arg Leu Pro Arg Asn Pro Lys Thr Asp

835 840 845

Asn Leu Val Asn His Arg Leu Ile Gly Met Ala Tyr Gly Gln Ile Gly

850 855 860

Met Ile Gln Ala Leu Ala Gly Phe Phe Thr Tyr Phe Val Ile Leu Ala

865 870 875 880

Glu Asn Gly Phe Arg Pro Val Asp Leu Leu Gly Ile Arg Leu His Trp

885 890 895

Glu Asp Lys Tyr Leu Asn Asp Leu Glu Asp Ser Tyr Gly Gln Gln Trp

900 905 910

Thr Tyr Glu Gln Arg Lys Val Val Glu Phe Thr Cys Gln Thr Ala Phe

915 920 925

Phe Val Thr Ile Val Val Val Gln Trp Ala Asp Leu Ile Ile Ser Lys

930 935 940

Thr Arg Arg Asn Ser Leu Phe Gln Gln Gly Met Arg Asn Lys Val Leu

945 950 955 960

Ile Phe Gly Ile Leu Glu Glu Thr Leu Leu Ala Ala Phe Leu Ser Tyr

965 970 975

Thr Pro Gly Met Asp Val Ala Leu Arg Met Tyr Pro Leu Lys Ile Thr

980 985 990

Trp Trp Leu Cys Ala Ile Pro Tyr Ser Ile Leu Ile Phe Val Tyr Asp

995 1000 1005

Glu Ile Arg Lys Leu Leu Ile Arg Gln His Pro Asp Gly Trp Val

1010 1015 1020

Glu Arg Glu Thr Tyr Tyr

1025

<210> 209

<211> 279

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 209

Met Thr Lys Asn Glu Lys Lys Ser Leu Asn Gln Ser Leu Ala Glu Trp

1 5 10 15

Lys Leu Phe Ile Tyr Asn Pro Thr Thr Gly Glu Phe Leu Gly Arg Thr

20 25 30

Ala Lys Ser Trp Gly Leu Ile Leu Leu Phe Tyr Leu Val Phe Tyr Gly

35 40 45

Phe Leu Ala Ala Leu Phe Ser Phe Thr Met Trp Val Met Leu Gln Thr

50 55 60

Leu Asn Asp Glu Val Pro Lys Tyr Arg Asp Gln Ile Pro Ser Pro Gly

65 70 75 80

Leu Met Val Phe Pro Lys Pro Val Thr Ala Leu Glu Tyr Thr Phe Ser

85 90 95

Arg Ser Asp Pro Thr Ser Tyr Ala Gly Tyr Ile Glu Asp Leu Lys Lys

100 105 110

Phe Leu Lys Pro Tyr Thr Leu Glu Glu Gln Lys Asn Leu Thr Val Cys

115 120 125

Pro Asp Gly Ala Leu Phe Glu Gln Lys Gly Pro Val Tyr Val Ala Cys

130 135 140

Gln Phe Pro Ile Ser Leu Leu Gln Ala Cys Ser Gly Met Asn Asp Pro

145 150 155 160

Asp Phe Gly Tyr Ser Gln Gly Asn Pro Cys Ile Leu Val Lys Met Asn

165 170 175

Arg Ile Ile Gly Leu Lys Pro Glu Gly Val Pro Arg Ile Asp Cys Val

180 185 190

Ser Lys Asn Glu Asp Ile Pro Asn Val Ala Val Tyr Pro His Asn Gly

195 200 205

Met Ile Asp Leu Lys Tyr Phe Pro Tyr Tyr Gly Lys Lys Leu His Val

210 215 220

Gly Tyr Leu Gln Pro Leu Val Ala Val Gln Val Ser Phe Ala Pro Asn

225 230 235 240

Asn Thr Gly Lys Glu Val Thr Val Glu Cys Lys Ile Asp Gly Ser Ala

245 250 255

Asn Leu Lys Ser Gln Asp Asp Arg Asp Lys Phe Leu Gly Arg Val Met

260 265 270

Phe Lys Ile Thr Ala Arg Ala

275

<210> 210

<211> 1258

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 210

Met Gly Asp Met Ala Asn Asn Ser Val Ala Tyr Ser Gly Val Lys Asn

1 5 10 15

Ser Leu Lys Glu Ala Asn His Asp Gly Asp Phe Gly Ile Thr Leu Ala

20 25 30

Glu Leu Arg Ala Leu Met Glu Leu Arg Ser Thr Asp Ala Leu Arg Lys

35 40 45

Ile Gln Glu Ser Tyr Gly Asp Val Tyr Gly Ile Cys Thr Lys Leu Lys

50 55 60

Thr Ser Pro Asn Glu Gly Leu Ser Gly Asn Pro Ala Asp Leu Glu Arg

65 70 75 80

Arg Glu Ala Val Phe Gly Lys Asn Phe Ile Pro Pro Lys Lys Pro Lys

85 90 95

Thr Phe Leu Gln Leu Val Trp Glu Ala Leu Gln Asp Val Thr Leu Ile

100 105 110

Ile Leu Glu Ile Ala Ala Ile Val Ser Leu Gly Leu Ser Phe Tyr Gln

115 120 125

Pro Pro Glu Gly Asp Asn Ala Leu Cys Gly Glu Val Ser Val Gly Glu

130 135 140

Glu Glu Gly Glu Gly Glu Thr Gly Trp Ile Glu Gly Ala Ala Ile Leu

145 150 155 160

Leu Ser Val Val Cys Val Val Leu Val Thr Ala Phe Asn Asp Trp Ser

165 170 175

Lys Glu Lys Gln Phe Arg Gly Leu Gln Ser Arg Ile Glu Gln Glu Gln

180 185 190

Lys Phe Thr Val Ile Arg Gly Gly Gln Val Ile Gln Ile Pro Val Ala

195 200 205

Asp Ile Thr Val Gly Asp Ile Ala Gln Val Lys Tyr Gly Asp Leu Leu

210 215 220

Pro Ala Asp Gly Ile Leu Ile Gln Gly Asn Asp Leu Lys Ile Asp Glu

225 230 235 240

Ser Ser Leu Thr Gly Glu Ser Asp His Val Lys Lys Ser Leu Asp Lys

245 250 255

Asp Pro Leu Leu Leu Ser Gly Thr His Val Met Glu Gly Ser Gly Arg

260 265 270

Met Val Val Thr Ala Val Gly Val Asn Ser Gln Thr Gly Ile Ile Phe

275 280 285

Thr Leu Leu Gly Ala Gly Gly Glu Glu Glu Glu Lys Lys Asp Glu Lys

290 295 300

Lys Lys Glu Lys Lys Asn Lys Lys Gln Asp Gly Ala Ile Glu Asn Arg

305 310 315 320

Asn Lys Ala Lys Ala Gln Asp Gly Ala Ala Met Glu Met Gln Pro Leu

325 330 335

Lys Ser Glu Glu Gly Gly Asp Gly Asp Glu Lys Asp Lys Lys Lys Ala

340 345 350

Asn Leu Pro Lys Lys Glu Lys Ser Val Leu Gln Gly Lys Leu Thr Lys

355 360 365

Leu Ala Val Gln Ile Gly Lys Ala Gly Leu Leu Met Ser Ala Ile Thr

370 375 380

Val Ile Ile Leu Val Leu Tyr Phe Val Ile Asp Thr Phe Trp Val Gln

385 390 395 400

Lys Arg Pro Trp Leu Ala Glu Cys Thr Pro Ile Tyr Ile Gln Tyr Phe

405 410 415

Val Lys Phe Phe Ile Ile Gly Val Thr Val Leu Val Val Ala Val Pro

420 425 430

Glu Gly Leu Pro Leu Ala Val Thr Ile Ser Leu Ala Tyr Ser Val Lys

435 440 445

Lys Met Met Lys Asp Asn Asn Leu Val Arg His Leu Asp Ala Cys Glu

450 455 460

Thr Met Gly Asn Ala Thr Ala Ile Cys Ser Asp Lys Thr Gly Thr Leu

465 470 475 480

Thr Met Asn Arg Met Thr Val Val Gln Ala Tyr Ile Asn Glu Lys His

485 490 495

Tyr Lys Lys Val Pro Glu Pro Glu Ala Ile Pro Pro Asn Ile Leu Ser

500 505 510

Tyr Leu Val Thr Gly Ile Ser Val Asn Cys Ala Tyr Thr Ser Lys Ile

515 520 525

Leu Pro Pro Glu Lys Glu Gly Gly Leu Pro Arg His Val Gly Asn Lys

530 535 540

Thr Glu Cys Ala Leu Leu Gly Leu Leu Leu Asp Leu Lys Arg Asp Tyr

545 550 555 560

Gln Asp Val Arg Asn Glu Ile Pro Glu Glu Ala Leu Tyr Lys Val Tyr

565 570 575

Thr Phe Asn Ser Val Arg Lys Ser Met Ser Thr Val Leu Lys Asn Ser

580 585 590

Asp Gly Ser Tyr Arg Ile Phe Ser Lys Gly Ala Ser Glu Ile Ile Leu

595 600 605

Lys Lys Cys Phe Lys Ile Leu Ser Ala Asn Gly Glu Ala Lys Val Phe

610 615 620

Arg Pro Arg Asp Arg Asp Asp Ile Val Lys Thr Val Ile Glu Pro Met

625 630 635 640

Ala Ser Glu Gly Leu Arg Thr Ile Cys Leu Ala Phe Arg Asp Phe Pro

645 650 655

Ala Gly Glu Pro Glu Pro Glu Trp Asp Asn Glu Asn Asp Ile Val Thr

660 665 670

Gly Leu Thr Cys Ile Ala Val Val Gly Ile Glu Asp Pro Val Arg Pro

675 680 685

Glu Val Pro Asp Ala Ile Lys Lys Cys Gln Arg Ala Gly Ile Thr Val

690 695 700

Arg Met Val Thr Gly Asp Asn Ile Asn Thr Ala Arg Ala Ile Ala Thr

705 710 715 720

Lys Cys Gly Ile Leu His Pro Gly Glu Asp Phe Leu Cys Leu Glu Gly

725 730 735

Lys Asp Phe Asn Arg Arg Ile Arg Asn Glu Lys Gly Glu Ile Glu Gln

740 745 750

Glu Arg Ile Asp Lys Ile Trp Pro Lys Leu Arg Val Leu Ala Arg Ser

755 760 765

Ser Pro Thr Asp Lys His Thr Leu Val Lys Gly Ile Ile Asp Ser Thr

770 775 780

Val Ser Asp Gln Arg Gln Val Val Ala Val Thr Gly Asp Gly Thr Asn

785 790 795 800

Asp Gly Pro Ala Leu Lys Lys Ala Asp Val Gly Phe Ala Met Gly Ile

805 810 815

Ala Gly Thr Asp Val Ala Lys Glu Ala Ser Asp Ile Ile Leu Thr Asp

820 825 830

Asp Asn Phe Thr Ser Ile Val Lys Ala Val Met Trp Gly Arg Asn Val

835 840 845

Tyr Asp Ser Ile Ser Lys Phe Leu Gln Phe Gln Leu Thr Val Asn Val

850 855 860

Val Ala Val Ile Val Ala Phe Thr Gly Ala Cys Ile Thr Gln Asp Ser

865 870 875 880

Pro Leu Lys Ala Val Gln Met Leu Trp Val Asn Leu Ile Met Asp Thr

885 890 895

Leu Ala Ser Leu Ala Leu Ala Thr Glu Pro Pro Thr Glu Ser Leu Leu

900 905 910

Leu Arg Lys Pro Tyr Gly Arg Asn Lys Pro Leu Ile Ser Arg Thr Met

915 920 925

Met Lys Asn Ile Leu Gly His Ala Phe Tyr Gln Leu Val Val Val Phe

930 935 940

Thr Leu Leu Phe Ala Gly Glu Lys Phe Phe Asp Ile Asp Ser Gly Arg

945 950 955 960

Asn Ala Pro Leu His Ala Pro Pro Ser Glu His Tyr Thr Ile Val Phe

965 970 975

Asn Thr Phe Val Leu Met Gln Leu Phe Asn Glu Ile Asn Ala Arg Lys

980 985 990

Ile His Gly Glu Arg Asn Val Phe Glu Gly Ile Phe Asn Asn Ala Ile

995 1000 1005

Phe Cys Thr Ile Val Leu Gly Thr Phe Val Val Gln Ile Ile Ile

1010 1015 1020

Val Gln Phe Gly Gly Lys Pro Phe Ser Cys Ser Glu Leu Ser Ile

1025 1030 1035

Glu Gln Trp Leu Trp Ser Ile Phe Leu Gly Met Gly Thr Leu Leu

1040 1045 1050

Trp Gly Gln Leu Ile Ser Thr Ile Pro Thr Ser Arg Leu Lys Phe

1055 1060 1065

Leu Lys Glu Ala Gly His Gly Thr Gln Lys Glu Glu Ile Pro Glu

1070 1075 1080

Glu Glu Leu Ala Glu Asp Val Glu Glu Ile Asp His Ala Glu Arg

1085 1090 1095

Glu Leu Arg Arg Gly Gln Ile Leu Trp Phe Arg Gly Leu Asn Arg

1100 1105 1110

Ile Gln Thr Gln Met Asp Val Val Asn Ala Phe Gln Ser Gly Ser

1115 1120 1125

Ser Ile Gln Gly Ala Leu Arg Arg Gln Pro Ser Ile Ala Ser Gln

1130 1135 1140

His His Asp Val Thr Asn Ile Ser Thr Pro Thr His Ile Arg Val

1145 1150 1155

Val Asn Ala Phe Arg Ser Ser Leu Tyr Glu Gly Leu Glu Lys Pro

1160 1165 1170

Glu Ser Arg Ser Ser Ile His Asn Phe Met Thr His Pro Glu Phe

1175 1180 1185

Arg Ile Glu Asp Ser Glu Pro His Ile Pro Leu Ile Asp Asp Thr

1190 1195 1200

Asp Ala Glu Asp Asp Ala Pro Thr Lys Arg Asn Ser Ser Pro Pro

1205 1210 1215

Pro Ser Pro Asn Lys Asn Asn Asn Ala Val Asp Ser Gly Ile His

1220 1225 1230

Leu Thr Ile Glu Met Asn Lys Ser Ala Thr Ser Ser Ser Pro Gly

1235 1240 1245

Ser Pro Leu His Ser Leu Glu Thr Ser Leu

1250 1255

<210> 211

<211> 1272

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 211

Met Gly Asp Met Thr Asn Ser Asp Phe Tyr Ser Lys Asn Gln Arg Asn

1 5 10 15

Glu Ser Ser His Gly Gly Glu Phe Gly Cys Thr Met Glu Glu Leu Arg

20 25 30

Ser Leu Met Glu Leu Arg Gly Thr Glu Ala Val Val Lys Ile Lys Glu

35 40 45

Thr Tyr Gly Asp Thr Glu Ala Ile Cys Arg Arg Leu Lys Thr Ser Pro

50 55 60

Val Glu Gly Leu Pro Gly Thr Ala Pro Asp Leu Glu Lys Arg Lys Gln

65 70 75 80

Ile Phe Gly Gln Asn Phe Ile Pro Pro Lys Lys Pro Lys Thr Phe Leu

85 90 95

Gln Leu Val Trp Glu Ala Leu Gln Asp Val Thr Leu Ile Ile Leu Glu

100 105 110

Ile Ala Ala Ile Ile Ser Leu Gly Leu Ser Phe Tyr His Pro Pro Gly

115 120 125

Glu Gly Asn Glu Gly Cys Ala Thr Ala Gln Gly Gly Ala Glu Asp Glu

130 135 140

Gly Glu Ala Glu Ala Gly Trp Ile Glu Gly Ala Ala Ile Leu Leu Ser

145 150 155 160

Val Ile Cys Val Val Leu Val Thr Ala Phe Asn Asp Trp Ser Lys Glu

165 170 175

Lys Gln Phe Arg Gly Leu Gln Ser Arg Ile Glu Gln Glu Gln Lys Phe

180 185 190

Thr Val Val Arg Ala Gly Gln Val Val Gln Ile Pro Val Ala Glu Ile

195 200 205

Val Val Gly Asp Ile Ala Gln Val Lys Tyr Gly Asp Leu Leu Pro Ala

210 215 220

Asp Gly Leu Phe Ile Gln Gly Asn Asp Leu Lys Ile Asp Glu Ser Ser

225 230 235 240

Leu Thr Gly Glu Ser Asp Gln Val Arg Lys Ser Val Asp Lys Asp Pro

245 250 255

Met Leu Leu Ser Gly Thr His Val Met Glu Gly Ser Gly Arg Met Leu

260 265 270

Val Thr Ala Val Gly Val Asn Ser Gln Thr Gly Ile Ile Phe Thr Leu

275 280 285

Leu Gly Ala Gly Gly Glu Glu Glu Glu Lys Lys Asp Lys Lys Gly Val

290 295 300

Lys Lys Gly Asp Gly Leu Gln Leu Pro Ala Ala Asp Gly Ala Ala Ala

305 310 315 320

Ser Asn Ala Ala Asp Ser Ala Asn Ala Ser Leu Val Asn Gly Lys Met

325 330 335

Gln Asp Gly Asn Val Asp Ala Ser Gln Ser Lys Ala Lys Gln Gln Asp

340 345 350

Gly Ala Ala Ala Met Glu Met Gln Pro Leu Lys Ser Ala Glu Gly Gly

355 360 365

Asp Ala Asp Asp Arg Lys Lys Ala Ser Met His Lys Lys Glu Lys Ser

370 375 380

Val Leu Gln Gly Lys Leu Thr Lys Leu Ala Val Gln Ile Gly Lys Ala

385 390 395 400

Gly Leu Val Met Ser Ala Ile Thr Val Ile Ile Leu Val Leu Tyr Phe

405 410 415

Thr Val Asp Thr Phe Val Val Asn Lys Lys Pro Trp Leu Pro Glu Cys

420 425 430

Thr Pro Val Tyr Val Gln Tyr Phe Val Lys Phe Phe Ile Ile Gly Val

435 440 445

Thr Val Leu Val Val Ala Val Pro Glu Gly Leu Pro Leu Ala Val Thr

450 455 460

Ile Ser Leu Ala Tyr Ser Val Lys Lys Met Met Lys Asp Asn Asn Leu

465 470 475 480

Val Arg His Leu Asp Ala Cys Glu Thr Met Gly Asn Ala Thr Ala Ile

485 490 495

Cys Ser Asp Lys Thr Gly Thr Leu Thr Thr Asn Arg Met Thr Val Val

500 505 510

Gln Ala Tyr Val Gly Asp Val His Tyr Lys Glu Ile Pro Asp Pro Ser

515 520 525

Ser Ile Asn Thr Lys Thr Met Glu Leu Leu Ile Asn Ala Ile Ala Ile

530 535 540

Asn Ser Ala Tyr Thr Thr Lys Ile Leu Pro Pro Glu Lys Glu Gly Ala

545 550 555 560

Leu Pro Arg Gln Val Gly Asn Lys Thr Glu Cys Gly Leu Leu Gly Phe

565 570 575

Val Leu Asp Leu Lys Gln Asp Tyr Glu Pro Val Arg Ser Gln Met Pro

580 585 590

Glu Glu Lys Leu Tyr Lys Val Tyr Thr Phe Asn Ser Val Arg Lys Ser

595 600 605

Met Ser Thr Val Ile Lys Leu Pro Asp Glu Ser Phe Arg Met Tyr Ser

610 615 620

Lys Gly Ala Ser Glu Ile Val Leu Lys Lys Cys Cys Lys Ile Leu Asn

625 630 635 640

Gly Ala Gly Glu Pro Arg Val Phe Arg Pro Arg Asp Arg Asp Glu Met

645 650 655

Val Lys Lys Val Ile Glu Pro Met Ala Cys Asp Gly Leu Arg Thr Ile

660 665 670

Cys Val Ala Tyr Arg Asp Phe Pro Ser Ser Pro Glu Pro Asp Trp Asp

675 680 685

Asn Glu Asn Asp Ile Leu Asn Glu Leu Thr Cys Ile Cys Val Val Gly

690 695 700

Ile Glu Asp Pro Val Arg Pro Glu Val Pro Glu Ala Ile Arg Lys Cys

705 710 715 720

Gln Arg Ala Gly Ile Thr Val Arg Met Val Thr Gly Asp Asn Ile Asn

725 730 735

Thr Ala Arg Ala Ile Ala Ile Lys Cys Gly Ile Ile His Pro Gly Glu

740 745 750

Asp Phe Leu Cys Leu Glu Gly Lys Glu Phe Asn Arg Arg Ile Arg Asn

755 760 765

Glu Lys Gly Glu Ile Glu Gln Glu Arg Ile Asp Lys Ile Trp Pro Lys

770 775 780

Leu Arg Val Leu Ala Arg Ser Ser Pro Thr Asp Lys His Thr Leu Val

785 790 795 800

Lys Gly Ile Ile Asp Ser Thr His Thr Glu Gln Arg Gln Val Val Ala

805 810 815

Val Thr Gly Asp Gly Thr Asn Asp Gly Pro Ala Leu Lys Lys Ala Asp

820 825 830

Val Gly Phe Ala Met Gly Ile Ala Gly Thr Asp Val Ala Lys Glu Ala

835 840 845

Ser Asp Ile Ile Leu Thr Asp Asp Asn Phe Ser Ser Ile Val Lys Ala

850 855 860

Val Met Trp Gly Arg Asn Val Tyr Asp Ser Ile Ser Lys Phe Leu Gln

865 870 875 880

Phe Gln Leu Thr Val Asn Val Val Ala Val Ile Val Ala Phe Thr Gly

885 890 895

Ala Cys Ile Thr Gln Asp Ser Pro Leu Lys Ala Val Gln Met Leu Trp

900 905 910

Val Asn Leu Ile Met Asp Thr Phe Ala Ser Leu Ala Leu Ala Thr Glu

915 920 925

Pro Pro Thr Glu Thr Leu Leu Leu Arg Lys Pro Tyr Gly Arg Asn Lys

930 935 940

Pro Leu Ile Ser Arg Thr Met Met Lys Asn Ile Leu Gly His Ala Val

945 950 955 960

Tyr Gln Leu Ala Leu Ile Phe Thr Leu Leu Phe Val Gly Glu Lys Met

965 970 975

Phe Gln Ile Asp Ser Gly Arg Asn Ala Pro Leu His Ser Pro Pro Ser

980 985 990

Glu His Tyr Thr Ile Ile Phe Asn Thr Phe Val Met Met Gln Leu Phe

995 1000 1005

Asn Glu Ile Asn Ala Arg Lys Ile His Gly Glu Arg Asn Val Phe

1010 1015 1020

Asp Gly Ile Phe Arg Asn Pro Ile Phe Cys Thr Ile Val Leu Gly

1025 1030 1035

Thr Phe Ala Ile Gln Ile Val Ile Val Gln Phe Gly Gly Lys Pro

1040 1045 1050

Phe Ser Cys Ser Pro Leu Gln Leu Asp Gln Trp Met Trp Cys Ile

1055 1060 1065

Phe Ile Gly Leu Gly Glu Leu Val Trp Gly Gln Val Ile Ala Thr

1070 1075 1080

Ile Pro Thr Ser Arg Leu Lys Phe Leu Lys Glu Ala Gly Arg Leu

1085 1090 1095

Thr Gln Lys Glu Glu Ile Pro Glu Glu Glu Leu Asn Glu Asp Val

1100 1105 1110

Glu Glu Ile Asp His Ala Glu Arg Glu Leu Arg Arg Gly Gln Ile

1115 1120 1125

Leu Trp Phe Arg Gly Leu Asn Arg Ile Gln Thr Gln Ile Glu Val

1130 1135 1140

Val Asn Thr Phe Lys Ser Gly Ala Ser Phe Gln Gly Ala Leu Arg

1145 1150 1155

Arg Gln Ser Ser Val Thr Ser Gln Ser Gln Asp Ile Arg Val Val

1160 1165 1170

Lys Ala Phe Arg Ser Ser Leu Tyr Glu Gly Leu Glu Lys Pro Glu

1175 1180 1185

Ser Arg Thr Ser Ile His Asn Phe Met Ala His Pro Glu Phe Arg

1190 1195 1200

Ile Glu Asp Ser Gln Pro His Ile Pro Leu Ile Asp Asp Thr Asp

1205 1210 1215

Leu Glu Glu Asp Ala Ala Leu Lys Gln Asn Ser Ser Pro Pro Ser

1220 1225 1230

Ser Leu Asn Lys Asn Asn Ser Ala Ile Asp Ser Gly Ile Asn Leu

1235 1240 1245

Thr Thr Asp Thr Ser Lys Ser Ala Thr Ser Ser Ser Pro Gly Ser

1250 1255 1260

Pro Ile His Ser Leu Glu Thr Ser Leu

1265 1270

<210> 212

<211> 874

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 212

Met Gly Asp Met Ala Asn Ser Ser Ile Glu Phe His Pro Lys Pro Gln

1 5 10 15

Gln Gln Arg Asp Val Pro Gln Ala Gly Gly Phe Gly Cys Thr Leu Ala

20 25 30

Glu Leu Arg Thr Leu Met Glu Leu Arg Gly Ala Glu Ala Leu Gln Lys

35 40 45

Ile Glu Glu Ala Tyr Gly Asp Val Ser Gly Leu Cys Arg Arg Leu Lys

50 55 60

Thr Ser Pro Thr Glu Gly Leu Ala Asp Asn Thr Asn Asp Leu Glu Lys

65 70 75 80

Arg Arg Gln Ile Tyr Gly Gln Asn Phe Ile Pro Pro Lys Gln Pro Lys

85 90 95

Thr Phe Leu Gln Leu Val Trp Glu Ala Leu Gln Asp Val Thr Leu Ile

100 105 110

Ile Leu Glu Val Ala Ala Ile Val Ser Leu Gly Leu Ser Phe Tyr Ala

115 120 125

Pro Pro Gly Glu Glu Ser Glu Ala Cys Gly Asn Val Ser Gly Gly Ala

130 135 140

Glu Asp Glu Gly Glu Ala Glu Ala Gly Trp Ile Glu Gly Ala Ala Ile

145 150 155 160

Leu Leu Ser Val Ile Cys Val Val Leu Val Thr Ala Phe Asn Asp Trp

165 170 175

Ser Lys Glu Lys Gln Phe Arg Gly Leu Gln Ser Arg Ile Glu Gln Glu

180 185 190

Gln Lys Phe Thr Val Ile Arg Asn Gly Gln Leu Leu Gln Val Pro Val

195 200 205

Ala Ala Leu Val Val Gly Asp Ile Ala Gln Val Lys Tyr Gly Asp Leu

210 215 220

Leu Pro Ala Asp Gly Val Leu Ile Gln Ala Asn Asp Leu Lys Ile Asp

225 230 235 240

Glu Ser Ser Leu Thr Gly Glu Ser Asp His Val Arg Lys Ser Ala Asp

245 250 255

Lys Asp Pro Met Leu Leu Ser Gly Thr His Val Met Glu Gly Ser Gly

260 265 270

Arg Met Val Val Thr Ala Val Gly Val Asn Ser Gln Thr Gly Ile Ile

275 280 285

Phe Thr Leu Leu Gly Ala Gly Gly Glu Glu Glu Glu Lys Lys Asp Lys

290 295 300

Lys Gly Lys Gln Gln Asp Gly Ala Met Glu Ser Ser Gln Thr Lys Ala

305 310 315 320

Lys Lys Gln Asp Gly Ala Val Ala Met Glu Met Gln Pro Leu Lys Ser

325 330 335

Ala Glu Gly Gly Glu Met Glu Glu Arg Glu Lys Lys Lys Ala Asn Ala

340 345 350

Pro Lys Lys Glu Lys Ser Val Leu Gln Gly Lys Leu Thr Lys Leu Ala

355 360 365

Val Gln Ile Gly Lys Ala Gly Leu Val Met Ser Ala Ile Thr Val Ile

370 375 380

Ile Leu Val Leu Tyr Phe Val Ile Glu Thr Phe Val Val Glu Gly Arg

385 390 395 400

Thr Trp Leu Ala Glu Cys Thr Pro Val Tyr Val Gln Tyr Phe Val Lys

405 410 415

Phe Phe Ile Ile Gly Val Thr Val Leu Val Val Ala Val Pro Glu Gly

420 425 430

Leu Pro Leu Ala Val Thr Ile Ser Leu Ala Tyr Ser Val Lys Lys Met

435 440 445

Met Lys Asp Asn Asn Leu Val Arg His Leu Asp Ala Cys Glu Thr Met

450 455 460

Gly Asn Ala Thr Ala Ile Cys Ser Asp Lys Thr Gly Thr Leu Thr Thr

465 470 475 480

Asn Arg Met Thr Val Val Gln Ser Tyr Leu Gly Asp Thr His Tyr Lys

485 490 495

Glu Ile Pro Ala Pro Ser Ala Leu Thr Pro Lys Ile Leu Asp Leu Leu

500 505 510

Val His Ala Ile Ser Ile Asn Ser Ala Tyr Thr Thr Lys Ile Leu Pro

515 520 525

Pro Glu Lys Glu Gly Ala Leu Pro Arg Gln Val Gly Asn Lys Thr Glu

530 535 540

Cys Ala Leu Leu Gly Phe Val Leu Asp Leu Lys Arg Asp Phe Gln Pro

545 550 555 560

Val Arg Glu Gln Ile Pro Glu Asp Lys Leu Tyr Lys Val Tyr Thr Phe

565 570 575

Asn Ser Val Arg Lys Ser Met Ser Thr Val Ile Arg Met Pro Asp Gly

580 585 590

Gly Phe Arg Leu Phe Ser Lys Gly Ala Ser Glu Ile Leu Leu Lys Lys

595 600 605

Cys Thr Asn Ile Leu Asn Ser Asn Gly Glu Leu Arg Gly Phe Arg Pro

610 615 620

Arg Asp Arg Asp Asp Met Val Arg Lys Ile Ile Glu Pro Met Ala Cys

625 630 635 640

Asp Gly Leu Arg Thr Ile Cys Ile Ala Tyr Arg Asp Phe Ser Ala Gly

645 650 655

Gln Glu Pro Asp Trp Asp Asn Glu Asn Glu Val Val Gly Asp Leu Thr

660 665 670

Cys Ile Ala Val Val Gly Ile Glu Asp Pro Val Arg Pro Glu Val Pro

675 680 685

Glu Ala Ile Arg Lys Cys Gln Arg Ala Gly Ile Thr Val Arg Met Val

690 695 700

Thr Gly Asp Asn Ile Asn Thr Ala Arg Ala Ile Ala Ala Lys Cys Gly

705 710 715 720

Ile Ile Gln Pro Gly Glu Asp Phe Leu Cys Leu Glu Gly Lys Glu Phe

725 730 735

Asn Arg Arg Ile Arg Asn Glu Lys Gly Glu Ile Glu Gln Glu Arg Leu

740 745 750

Asp Lys Val Trp Pro Lys Leu Arg Val Leu Ala Arg Ser Ser Pro Thr

755 760 765

Asp Lys His Thr Leu Val Lys Gly Ile Ile Asp Ser Thr Thr Gly Glu

770 775 780

Gln Arg Gln Val Val Ala Val Thr Gly Asp Gly Thr Asn Asp Gly Pro

785 790 795 800

Ala Leu Lys Lys Ala Asp Val Gly Phe Ala Met Gly Ile Ala Gly Thr

805 810 815

Asp Val Ala Lys Glu Ala Ser Asp Ile Ile Leu Thr Asp Asp Asn Phe

820 825 830

Thr Ser Ile Val Lys Ala Val Met Trp Gly Arg Asn Val Tyr Asp Ser

835 840 845

Ile Ser Lys Phe Leu Gln Phe Gln Leu Thr Val Asn Val Val Ala Val

850 855 860

Ile Val Ala Phe Thr Gly Ala Cys Ile Thr

865 870

<210> 213

<211> 28

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 213

Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr

1 5 10 15

Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys

20 25

<210> 214

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 214

Thr Val Phe Leu Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro

1 5 10 15

Lys Arg

<210> 215

<211> 415

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 215

Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg

1 5 10 15

Glu Cys Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe

20 25 30

Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly

35 40 45

Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp

50 55 60

Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys

65 70 75 80

Asn Cys Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu

85 90 95

Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr

100 105 110

Glu Gly Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val

115 120 125

Pro Phe Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr

130 135 140

Arg Ala Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu

145 150 155 160

Ala Glu Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn

165 170 175

Asp Phe Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe

180 185 190

Pro Trp Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly

195 200 205

Ser Ile Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu

210 215 220

Thr Gly Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu

225 230 235 240

Thr Glu His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His

245 250 255

His Asn Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu

260 265 270

Leu Glu Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile

275 280 285

Cys Ile Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser

290 295 300

Gly Tyr Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala

305 310 315 320

Leu Val Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys

325 330 335

Leu Arg Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly

340 345 350

Phe His Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro

355 360 365

His Val Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser

370 375 380

Trp Gly Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys

385 390 395 400

Val Ser Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr

405 410 415

<210> 216

<211> 415

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 216

Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg

1 5 10 15

Glu Cys Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe

20 25 30

Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly

35 40 45

Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp

50 55 60

Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys

65 70 75 80

Asn Cys Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu

85 90 95

Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr

100 105 110

Glu Gly Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val

115 120 125

Pro Phe Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr

130 135 140

Arg Ala Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu

145 150 155 160

Ala Glu Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn

165 170 175

Asp Phe Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe

180 185 190

Pro Trp Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly

195 200 205

Ser Ile Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu

210 215 220

Thr Gly Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu

225 230 235 240

Thr Glu His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His

245 250 255

His Asn Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu

260 265 270

Leu Glu Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile

275 280 285

Cys Ile Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser

290 295 300

Gly Tyr Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala

305 310 315 320

Leu Val Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys

325 330 335

Leu Leu Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly

340 345 350

Phe His Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro

355 360 365

His Val Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser

370 375 380

Trp Gly Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys

385 390 395 400

Val Ser Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr

405 410 415

<210> 217

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(8)

<223> (GGS) n (GGGGS) n-linker, wherein n is

An integer between 1 and 100

<400> 217

Gly Gly Ser Gly Gly Gly Gly Ser

1 5

<210> 218

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 218

Ser Gly Gly Ser Gly Gly Ser

1 5

<210> 219

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 219

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gly

1 5 10 15

<210> 220

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 220

Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 221

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 221

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

1 5 10 15

Gly Ser

<210> 222

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 222

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 223

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(4)

<223> (GGGG) n shortest example of linker, where n is

An integer between 1 and 100

<400> 223

Gly Gly Gly Gly

<210> 224

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X = any amino acid

<400> 224

Met Gly Xaa Lys Leu Ser Lys Lys Lys

1 5

<210> 225

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X = is any amino acid

<400> 225

Gly Xaa Lys Leu Ser Lys Lys Lys

1 5

<210> 226

<211> 22

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 226

ugagaacuga auuccauggg uu 22

<210> 227

<211> 22

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 227

ccucugaaau ucaguucuuc ag 22

<210> 228

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 228

uuaaugcuaa ucgugauagg ggu 23

<210> 229

<211> 22

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence

<400> 229

cuccuacaua uuagcauuaa ca 22

<210> 230

<211> 95

<212> PRT

<213> Mycobacterium tuberculosis (Mycobacterium tuberculosis)

<400> 230

Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser

1 5 10 15

Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly

20 25 30

Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser

35 40 45

Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu

50 55 60

Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly

65 70 75 80

Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala

85 90 95

<210> 231

<211> 96

<212> PRT

<213> Mycobacterium tuberculosis (Mycobacterium tuberculosis)

<400> 231

Met Ser Gln Ile Met Tyr Asn Tyr Pro Ala Met Leu Gly His Ala Gly

1 5 10 15

Asp Met Ala Gly Tyr Ala Gly Thr Leu Gln Ser Leu Gly Ala Glu Ile

20 25 30

Ala Val Glu Gln Ala Ala Leu Gln Ser Ala Trp Gln Gly Asp Thr Gly

35 40 45

Ile Thr Tyr Gln Ala Trp Gln Ala Gln Trp Asn Gln Ala Met Glu Asp

50 55 60

Leu Val Arg Ala Tyr His Ala Met Ser Ser Thr His Glu Ala Asn Thr

65 70 75 80

Met Ala Met Met Ala Arg Asp Pro Ala Glu Ala Ala Lys Trp Gly Gly

85 90 95

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